Caracterização bioquímica de proteases digestivas em larvas de Alphitobius diaperinus (Coleoptera: Tenebrionidae)

Abstract

Alphitobius diaperinus is considered an important issue in the avian environment. Studying your behavior, eating habits and digestive enzymes may indicate important targets in your control. Control of A. diaperinus is considered difficult, its natural enemies are little known, and the use of chemical insecticides is the main method of control. Insect digestive proteases catalyze the production of peptides and amino acids from a protein diet and they are found in the midgut region of insects. Proteolytic activities in the digestive tract of A. diperínus larvae and adults were determined using azocasein and specific substrates after feeding on different diets. The crude extract of the digestive tract of A. diaperinus larvae showed activity on azocasein and Abz-Phe-Arg-MCA. It was inhibited by more than 50% by E-64 and activated by 2.0 mM DTT at acid pH. Nine bands were detected with proteolytic activity and molecular mass in the range 10 kDa - 100 kDa through zymogram, using 0.1% gelatin as substrate. Different classes of proteases were identified in the presence of classical protease inhibitors such as E-64, iodoacetamide and iodoacetic acid, PMSF, SBTI, aprotinin and TLCK. A higher level of proteolytic activity was observed at pHs between 5 and 6. Although combinations of serine and cysteine proteases have been observed, acid proteases showed higher levels of proteolytic activity. Calcium ion increased the protease activity at concentrations greater than 2.0 mM. Two-dimensional nano-HPLC and ESI-MS / MS analyzes resulted in the identification of proteases such as tripine, cathepsins, metalloproteases, aminopeptidases, peptidases and dipeptidases. High inhibitory potential of different series of imidazolone on cysteine protease was observed, what can be used as control tools of A. diaperinus.