A number of 100 individuals of E. integriceps were used in this study. Female adults of E. integriceps were obtained from laboratory cultures. Insects were placed in containers (plastic dishes) with access to water and without feeding, in laboratory conditions at 25 ± 2 °C and a photoperiod of 14:10 (L: D) for 10 days. Then, the starved insects were allowed to feed on wheat kernels for 3 hours Then, a number of 20 insects were selected and dissected as described in the following section. Starved insects (20 insects) were also dissected and considered as controls.

Adult females were individually immobilized by placing them on crushed ice (8–10 min), then they were dissected under the stereomicroscope in pre-cold 215 mM NaCl, and their SGC were removed. Drawings were made with the aid of a stereomicroscope at 10× magnification.

Enzyme preparation was based on the previously described methods, with slight modifications [15,16]. Groups of pooled SGC were homogenized in distilled water by a motorized Potter–Elvehjem homogenizer (Teflon pestle, 0.1 mm clearance). Then, the SGC homogenate from starved and fed insects were transferred separately into 1.5-mL centrifuge tubes and centrifuged at 15,000 g for 30 min at 4 °C. The final volumes of both supernatant and pellet were adjusted to 1 mL by double-distilled water.

Protein concentration was measured according to the method of Bradford [17], using bovine serum albumin (Bio-Rad, Munich, Germany) as a standard.

The α-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure [18], using 1% soluble starch or 1% soluble glycogen. α- and β-glucosidase activities were measured using 5 mM of 4-nitrophenyl α-d-glucopyranoside and 4-nitrophenyl β-d-glucopyranoside (NPα/βGlu), respectively, in a 50 mM citrate–phosphate buffer at pH 5.0, based on the appearance of p-nitrophenol in the solution according to Terra et al. α-/β-Galactosidase activities were assessed using 5 mM of 4-nitrophenyl α-d-glucopyranoside and 4-nitrophenyl β-d-glucopyranoside (NPα/βGal), respectively, in a 50 mM citrate–phosphate buffer at pH 5.0. α- and β-manosidase activities were measured using 2.5 mM of 4-nitrophenyl α-d-manopyranoside and 4-nitrophenyl β-d-manopyranoside (NPα/βMan), respectively in a 50 mM citrate–phosphate buffer at pH 5.0. Lipase activity was measured using 1 mM of p-nitrophenyl butyrate (pNPB) in a 50 mM citrate–phosphate buffer at pH 5.0. Aminopeptidase activity was measured using 2 mM l-leucine p-nitroanilide (LpNA) in 50 mM of a Tris–HCl buffer at pH 7.0 as a substrate [19]. The activity of acid phosphatase was determined using 5 mM of 4-nitrophenyl phosphate (pNPP) in 50 mM of a citrate–phosphate buffer at pH 4.5 as a substrate. Alkaline phosphatase was determined using 50 mM of a glycine–NaOH mixture at pH 10.4 containing 1 mM of MgSO₄ [20]. General proteolysis was done according to the methods of Elpidina et al. [21] and Gatehouse et al. [22] using 2% azocasein in a Tris–HCl buffer at pH 8.0. Trypsin- and chymotrypsin-like activities were assayed using 1 mM of N-benzoyl-l-arginine-p-nitroanilide (BApNA) and 1 mM of N-succinyl-l-Ala-l-Ala-l-Pro-Phe-p-nitroanilide (SAAPFpNA) [21,22]. The substrate was dissolved in dimethyl sulfoxide (DMSO) and then diluted in a buffer so that the final concentration of DMSO in solution was less than 10%.

The hydrolysis of the substrates was monitored continuously at 410 nm at 30 °C and initial rates were measured from the slopes of absorbance against time. For each determination, the mixture of the reagents was kept at 30 °C for at least four different periods of time, and initial velocities were calculated. Controls without enzyme or substrate were carried out and treated in a similar way to the experimental groups. The enzyme activity was expressed in milli units (mU). One unit of enzymatic activity was defined as the amount of enzyme that hydrolyzes 1 mmol of substrate per minute.

The pH optima of enzymes were determined using a universal buffer [23]. The pH values tested were 3, 4, 5, 6, 7 and 8.

The dependence of the hydrolytic activity of the amylase on substrate concentration was analyzed. The assay was performed in the presence of substrate concentrations of 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8% of starch or glycogen, but the enzyme concentration was kept constant. Controls were run in parallel, in which distilled water replaced the enzyme for each substrate concentration. Double reciprocal plots (Lineweaver–Burk plots) of enzyme activity versus substrate concentration were used to establish that Michaelis–Menten kinetics were obeyed over this substrate concentration range and to obtain values of the maximum velocity (V_max) and Michaelis constant (K_M).

The crude extract (40 mL) from 100 salivary glands homogenates of E. integriceps adults was treated with ammonium sulfate at 4 °C to give fractions precipitating at 20%, 30%, 40%, 50%, 60%, and 80% saturation. The precipitates were collected by centrifugation at 10,000 g for 15 min, diluted in 2 mL of a 0.02 mol/L phosphate buffer (pH 6.5) and dialyzed overnight at 4 °C against the same buffer. The enzyme solution was applied to a Sephadex G-75 column (1.5 cm × 80 cm), equilibrated with a 0.02 mol/L phosphate buffer (pH 6.5). The column was run at a flow rate of 0.5 mL/min and 1.5 mL fractions were collected. The protein level of each fraction was monitored at 280 nm and amylase activity was measured as described in the previous section. Fractions with enzymatic activity were pooled and concentrated by lyophilization. The lyophilized powder was dissolved in the least amount of phosphate buffer and applied to a diethylaminoethyl (DEAE)-cellulose column (1.3 × 28 cm), equilibrated with a 0.02 mol/L phosphate buffer (pH 6.5). The enzyme elution was done at a flow rate of 0.5 mL/min with a linear NaCl gradient (0–0.6 mol/L). Fractions (1.0 mL/tube) were collected and their protein concentration and α-amylase activity were determined as described.

To detect the amylolytic activity of SGC, native SDS-PAGE was carried out. Native PAGE was performed in 10% (w/v) gel with 0.05% SDS for separating the gel from the stacking gel (5%) with 0.05% SDS. After electrophoresis, the gel was rinsed with distilled water and washed by gentle shaking in 1% (v/v) Triton X-100 dissolved in a phosphate buffer containing 2 mM of CaCl₂ and 10 mM of NaCl for 30 min Then the gel was rinsed with distilled water and treated with a 1% starch solution for 1.5 h. Finally, the gel was treated with a solution of 1.3% I₂, 3% KI to stop the reaction and to stain the unreacted starch background. Zones of α-amylase activities appeared as a light band against the dark background of the gel.

To have an insight into the protein contents of SGC, SDS-PAGE was applied. The acrylamide concentration was 10% for the separating gel and 4% for the stacking gel, and proteins on the polyacrylamide gel were stained with 0.2% of Coomassie brilliant blue R-250. β-Galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35.5 kDa), restriction endonuclease Bsp 981 (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) were used as molecular mass standards.

Data were compared by one-way analysis of variance (ANOVA) followed by LSD test. When P < 0.01, the differences were considered to be significant. To compare means of enzymatic activities before and after feeding, a paired-Student's t-test was also used.

The SCGs of E. integriceps occur in pairs, just around the digestive canal, and the glands extend back into the thorax. The SGCs consist of branching short tubules fusing together at the center of the salivary gland, producing concentric semicircles. Each tubule usually divides and forms a branched structure (Fig. 1). The glands are bathed in the hemocoel. The SGCs are composed of two sections, the principal gland (PG) and the accessory gland (AG) (Fig. 1). The principal glands are further divided into two lobes, which are referred to as anterior lobe (AL) and posterior lobe (PL). The shape of the posterior and anterior lobes is the same; the posterior lobe, however, is about 2 times bigger than the anterior one and is the main SGC. The accessory gland is an elongate tubular structure, fusing to the principal glands. The principal and accessory glands have their own ducts and these ducts lead to the lateral salivary duct. The lateral ducts from each side fuse to form a median salivary duct. The tracheoles are quite extended on the salivary glands surface. The salivary glands were reported to be acidic using chemical indicators (pH: 5.5–6.0) [7]. There was no difference in pH between salivary glands from starved and fed insects using chemical indicators (data not shown).

The extents of the enzymatic activities of SGC were dependent on the pH value of the assay medium (Fig. 2). Despite α-glucosidase with an optimum pH at 8.0, the highest carbohydrase activities were seen at pH 5.0–6.0 (Fig. 2). The activities dropped markedly outside the narrow optimum pH at 30 °C. Lipase had an optimum activity at pH values between 7.0 and 8.0, while phosphatase showed two pH optimum activities at 5.0 and 9.0 (Fig. 2).

Investigation of the profile of digestive enzymes of the salivary glands using general and specific substrates evidenced the major enzymatic activities in PG (especially PL) (data not shown). Activities of some enzymes in PL, however, were not high enough to be detected readily; then, whole SGC extract was used. Enzymatic activities against almost all the substrates used were found (Fig. 3). Results showed that salivary α-amylase had higher activity against starch than glycogen (Fig. 3A). Carbohydrases were detected in SGC with different activities; the largest carbohydrase activities were recorded for α/β-glucosidases. Enzymatic activities toward mannose (α/β-mannosidases) were low in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases) (Fig. 3B–D). Both alkaline and acid phosphatases were detected in SGC using buffers with different pHs. However, acid phosphatase showed a seven-fold higher activity than alkaline phosphatase (Fig. 3E). We detected no measurable activities for lipase and aminopeptidase. Proteolytic activity against hemoglobin was higher than that against azocasein (Fig. 3F). Moreover, trypsin activity was higher than chymotrypsin activity in the salivary glands, as detected by specific substrates (Fig. 3G).

To test the hypothesis that enzymatic activities were changed in response to starvation and feeding, investigation was done in starved and fed insects. The results showed that the activities of almost all enzymes were increased in response to feeding in comparison to starved insects (Fig. 3). Amylolytic activity against starch was increased significantly (t = 49.13, P < 0.01), while an increase in the activity against glycogen was not significant in fed insects (Fig. 3A). Despite α-glucosidase and α-mannosidase which induced significant responses to feeding (t = 37.76, P = 0.017 and t = 16.2, P < 0.05, respectively), enhanced carbohydrase activities in fed insects were not significantly higher than in starved insects (Fig. 3B–D). Acid phosphatase showed significantly higher activity in fed insects in comparison to the starved ones (t = 15.4, P < 0.05); alkaline phosphatase, however, did not change significantly (Fig. 3E). Proteolytic activities (general and specific) were increased in response to feeding, although these were not significant (Fig. 3F, G).

In complement to colorimetric assays, the SGC extract was subjected to a series of PAGE. We tested the induction of enzymatic activities in response to feeding with SDS-PAGE by using equal volumes from protein extracts of salivary glands pooled from starved and fed insects. Results showed that the intensities of the respective bands in the protein profile of the SGC of fed insects were enhanced in comparison with the starved insects’ SGC protein profile. Moreover, some new bands (less than 25 kDa) were detected only in the protein profile of the SGC of the fed insects (Fig. 4). It is noteworthy that induction of digestive amylase and protease in response to feeding was not high enough to be detected by native PAGE.

Considering the presence of a high α-amylase activity in SGC and also its prominent role in digestion in insects, α-amylases of SGC from E. integriceps adults were fractionated using gel filtration chromatography (Fig. 5). Each fraction was tested for amylolytic activity against starch 1% as a substrate. Fractions with high amylolytic activities were pooled as indicated in the chromatogram and subjected to PAGE analysis using the same substrate.

The SGC amylase activity eluted in four major peaks (noted as I to IV). The highest amylolytic activity in SGC fractions was observed in the first peak of the chromatogram with pH optima at 5.0 (Fig. 5A, C). The second peak of amylase activity showed an optimal pH value of 6.0 (Fig. 5A, C). Amylases in two other peaks (III and IV) from SGC had their maximal activities at pH values of 8.0 and 6.0, respectively (Fig. 5A, C).

In complement to colorimetric inhibition assays, the fractions were subjected to a series of non-denaturing PAGE. The post-electrophoretic detection of the starch digestion by amylase demonstrated existence of at least five isoamylases in the SGC (e.g., AMYa, AMYb, AMYc, AMYd, and AMYe) (Fig. 5B). Zymography of the four amylase-rich fractions of SGC showed that they contained different isoamylases (Fig. 5B). Since pooled fractions showed various optimum pH values as well as different isoamylases, some kinetic parameters were determined for the fractions. The results revealed that the fractions had different kinetic parameters against starch (1%) and glycogen (1%). Interestingly, it was shown that the amylases of the insect had more efficiency (e.g., V_max/K_M ratios) on starch than on glycogen (Table 1). There were significant differences among the catalytic parameters of the different fractions. Among the fractions, fraction IV and III showed the highest V_max/K_M ratios against starch and glycogen, respectively (Table 1).