We use only adult males, since preliminary studies using both male and females, resulted Sotrastaurin supplier in large variation in enzymes activities, probably due to physiological reproductive

variations in females. The insects were starved for 48 h and then fed ad libitum for 24 h with pupae of T. molitor L. Adults of P. nigrispinus were immobilized in cold and dissected in saline solution (0.1 M NaCl, 0.1 M KH2PO4, 0.1 M Na2HPO4, pH 7.2). Salivary glands and midguts were removed and stored at −80 °C until use. In some insects, the midgut was divided into three regions (anterior, middle and posterior). Samples of the salivary glands, whole midguts and midgut sections were homogenized in cold MilliQ water with the aid of a Potter–Elvehjem homogenizer. The homogenates were centrifuged at 16,000g for 30 min at 4 °C. The pellets and supernatants were stored at −20° C until use. For the enzymes assays pools of ten midguts were homogeneized in 500 μL of MiliQ water and 20 salivary glands in 100 μL in MiliQ water, whereas for enzymes purification a pool of 40 midguts were homogeneized in 1 mL of MiliQ water. No enzyme inactivation was detected on storage. The contents of the salivary glands and the midgut sections were dispersed in 5 μL PI3K Inhibitor high throughput screening of MilliQ water and added to 5 μL of a 5-fold dilution of a universal pH indicator (E. Merck,

Darmstadt, pH 4–10). The resulting colored solutions were compared with suitable standard solutions diluted in 5 μL of MilliQ water. Protein content in extracts was determined according to Smith et al. (1985) as modified by Morton and Evans (1992), using bovine serum albumin (BSA) as a standard. Unless otherwise specified, hydrolase

assays were performed as follows. α-Amylase activity was measured by determining the appearance of reducing groups (Noelting and Bernfeld, 1948) in 50 mM citrate–phosphate buffer at pH 6.0 using 0.5% (w/v) starch as substrate. Absorbance was measured at 550 nm. Aminopeptidase assays were accomplished using 1 mM l-leucine p-nitroanilide (LpNA) as substrate in 50 mM citrate–phosphate buffer pH 6.0, according to Erlanger et al. (1961) and absorbance measured at 550 nm. α-Glucosidase ifoxetine activity was determined by following the release of p-nitrophenolate from 5 mM p-nitrophenyl-α-d-glucoside (pNPαGlu) in 50 mM citrate–phosphate buffer pH 6.0 and absorbance measured at 420 nm, as described in Terra et al. (1979). Serine protease (trypsin and chymotrypsin) assays were performed in 0.1 M Tris–HCl, pH 7.5 as follows. Activities were quantified by determining the methyl-coumarin fluorescence (excitation 360 nm and emission 460 nm) released from 1 mM carbobenzoxy-Phe-Arg-7-amino-4-methylcoumarin (Z-FR-MCA) in the case of trypsin and 1 mM succinyl-Ala-Ala-Pro-Phe-7-amino-4-methyl-coumarin (Suc-AAPF-MCA) in the case of chymotrypsin.