Insect midgut proteases are excellent targets for insecticidal agents such as protease inhibitors. These inhibitors are used for producing transgenic plants, resistant to pests. For achieving this goal, it is necessary to find the nature of specific proteases and their properties for adopting possible pest management procedure. Therefore, characterisation of the enzymes in the gut of the rose sawfly, Arge rosae (Hymenoptera: Argidae), responsible for proteolysis, was performed using a range of synthetic substrates and specific inhibitors. The optimum conditions for general proteases and trypsin were achieved at pH 10. The highest activity for general proteases was obtained at a temperature of 458C. The use of specific inhibitors and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) provided evidence to suggest that most of the proteases belonged to the serine group because of high inhibitory effect of phenyl methane sulfonyl fluoride on total proteolytic activity. Also, inhibition assays and zymogram analysis showed that metalloproteases are present in A. rosae digestive system. These results indicated that A. rosae larvae mainly used serine proteases for protein digestion, with chymotrypsin as the dominant form. The kinetic parameters of trypsin-like proteases using N-benzoyl-DL-arg-p-nitroanilide as substrate indicated that the K m and V max values of trypsin in the gut of the fifth instar larvae were 730 + 17.3 mM and 456 + 13.85 nmol min 71 mg 71 protein, respectively.

Abstract:It is necessary to study the biochemical changes in insects exposed to toxicants if we want to predict the potential of various chemicals on the natural enemy. Physiological energy, as a biochemical biomarker, may be affected by many pesticides including organophosphate compounds. Therefore, in this study, the sublethal effects of diazinon, fenitrothion, and chlorpyrifos on the cellular energy allocation (CEA) of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae), a potential biological control agent, was studied on 5th-instar nymphs. Among the energy reserves of the A. spinidens nymphs, only total protein was significantly affected by pesticide treatments, and the highest value was observed in chlorpyrifos treatment. The energy available (E a ) and energy consumption (E c ) in A. spinidens were significantly affected by these pesticides. In exposed bugs, these parameters were affected by fenitrothion and chlorpyrifos more than diazinon. The activity of the electron transport system (ETS) in the Ec assay showed that A. spinidens exposed to chlorpyrifos had the highest rate of oxygen consumption. Although, there was no significant change in CEA, the insecticides caused a marked change in the physiological balance of A. spinidens. The results suggested that the adverse effect of these insecticides on A. spinidens should be considered in Integrated Pest Management (IPM) programs.

The biochemical properties of proteases from the digestive system of the fig tree skeletonizer moth, Choreutis nemorana, were determined. Gut extracts of C. nemorana larvae were analysed using different specific peptide substrates and proteinase inhibitors. The optimal pH and temperature for proteolytic activities using azocasein as substrate were obtained as pH 11 and 45°C, respectively. In the case of N-benzoyl-l-arg-p-nitroanilide as substrate, the enzyme showed the maximum tryptic activity at pH 11. The kinetic parameters of trypsin-like proteases indicated that the K m and V max values of trypsin in the gut of C. nemorana were 0.157 ± 0.006mM and 0.188 ± 0.005 µmol/min/mg protein. Using specific proteolytic inhibitors, the inhibitors including phenyl methane sulfonyl fluoride, N-p-tosyl-l-lys chloromethyl ketone and ethylene diamine tetraacetic acid showed the greatest inhibitory effect on total proteolytic activity. These results indicated that serine proteinases accounted for the major proteases in the gut of C. nemorana. Inhibition assays and zymogram analysis showed that only small amounts of cysteine proteases are present in the digestive system of C. nemorana.

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