Introduction
The pomegranate, Punica granatum L. (Punicaceae), is one of the oldest cultivated species which is native to Iran but nowadays it is widespread throughout the Mediterranean area of Asia, Africa and Europe (Durgac et al., 2008). The carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae), is one of the most important pest of stored products and fruit trees throughout the world (Nay and Perring, 2006). Thus, it is a major pest on date in Southern California (USA), almonds in Israel, pomegranate in Iraq and Pomegranate and pistachio in Iran (Gothilf, 1984; Warner, 1988; Behdad, 2002; Nay and Perring, 2005; Karami et al., 2011).

The larvae feed inside the fruit and cause great damages to fruit quality so that in some area it spoils more than 80% of the fruits (Shakeri, 2004). Various agricultural and mechanical methods including removing the flags, covering Pomegranate fruit neck by straw, collecting and destroying of the infested fruit during the winter season as well as biological control are listed for the pest control (Kishani et al., 2012; Nasrollahi et al., 1998). However, these control methods were not successful. Since the larvae feed inside the fruit, on one hand pesticide applications have not been successful and on the other hand pesticide use has its own limitation due to problem associated with their use. In addition, the public and academia concerns over the use of pesticides on fruits and food safety have raised many issues. In addition, the occurrence of resistance in the insect pests is another issue which strengthens the need for alternative pest control strategies in IPM (integrated pest management) (Hagstrum and Subramanyam, 1996; Yildirim et al., 2001; Isman, 2006). In recent years considerable investigations have been done on plants and microbes derived materials for potentially useful products and genes to be used in pest control. Many plants have received gene/s that encoding toxic proteins as a strategy to resist or be protected from insect pests and pathogens. Genes that their products are toxic to insect species and confer enhance resistance against pest that so far have been received great attention are lectins (Gatehouse et al., 1997), α-amylase inhibitors (Mehrabadi et al., 2012; Mehrabadi and Bandani, 2011), protease inhibitors (Saadatia et al., 2011), toxins from Bacillus thuriengiensis (Bt toxins) (Sharma and Ortiz, 2000), and even fusion proteins consisting of plant lectin, Galanthus nivalis agglutinin (GNA) linked to toxic peptide (Fitches et al., 2004; Down et al., 2006; Fitches et al., 2009).

Digestive system of insects is a good target for implementing control mechanisms that are not toxic to other organisms (Nauen et al., 2001). A large number of proteinaceous and non-proteinaceous molecules in plants act as part of the plant's natural defense against herbivores in order to prevent herbivors feeding (Mendiola-Olaya et al., 2000; Franco et al., 2002; Piasecka-Kwiatkowska et al., 2007). These inhibitors are widely existed in various plant seeds and tubers with greater abundance, especially in cereals and legumes (Iulek et al., 2000; Svensson et al., 2004; Bonavides et al., 2007; Mehrabadi et al., 2010). Thus, one important aspect of the insect pest control is to materialize selective inhibition of the digestive enzymes thus to interfere in digestion process of the insect by producing detrimental effects on larval and insect growth by inhibition of the digestion and assimilation of nutrients. Therefore, in order to achieve a control strategy based on digestive enzyme inhibitors, it is advisable to characterize digestive enzymes as well as to do in vitro and in vivo bioassays with plant proteinaceous inhibitors (Horrison and Bonning, 2010).

A first example of introduction of plant genes encoding toxic protein against insect is cowpea trypsin inhibitor which expressed in tobacco leaves to combat lepidopteran larvae (Hilder et al., 1987, Silva et al., 2001). Since then, attempts have been made to use toxic plant proteins against insects with some success. α-Amylase inhibitor gene from seeds of common bean (Phaseolus vulgaris) when transferred to pea confer resistance to pea weevil (Bruchus pisorum) (Morton et al., 2000; Silva et al., 2001). Also, transformed Azuki bean (Vigna angularis) confers resistance to Callosobruchus chinensis and C. Maculates (Ishimoto et al., 1996; Silva et al., 2001).

So far, no investigation has been done on the effect of plant origin toxic metabolites on the carob moth gut digestive enzymes. Thus, the aim of the current study was to investigate the effect of cereal seed proteinaceous extract including wheat cultivars and triticale against the insect gut digestive enzymes using spectrophotometric and in gel assays procedures.

2 Results
2.1 Determination of enzymes activity in different parts of the midgut
The α-amylase enzyme activity in the anterior, mid and posterior was 0.013 U/min/mg protein, 0.018 U/min/mg protein and 0.016 U/min/mg protein, respectively (Figure 1 and Figure 2A). Fgiure 2B shows α-amylase activity in the gel assay that two α-amylase bands are seen in the insect gut, one is A1 which is the major α-amylase band and the other is A2 with minor activity.

Figure 1 Different part of the carob moth midgut

Figure 2 A: Spectrophotometric amylase assay of three parts of the midgut; B: In gel assay of the α-amylase activity in three parts of the midgut including anterior (A), mid (M) and posterior (P) midgut

The proteolytic activity of the three gut parts including anterior, mid and posterior gut was 0.003 U/min, 0.004 U/min and 0.003 U/min, respectively (Figure 3A). As shown in figure 3B, there are two protease bands in the anterior, mid and posterior part of the midgut. However, Bands of mid part were sharper than the anterior and posterior midgut showing higher activity of these proteases in the middle part of the midgut (Figure 3B). These data showed that in the both enzymes (α-amylase and protease), activities were more in the midsection of the midgut than those of the posterior and anterior midgut. 

Figure 3 A: Spectrophotometric protease assays of the three parts of the midgut; B: in gel assays of the protease activity in three parts of the midgut including anterior (A), mid (M) and posterior (P) midgut

2.2 Effects of the seed extracts on the α-amylase activity
α-amylase activity in the presence of wheat and triticale seed extracts showed a dose dependent inhibition (Figure 4). The effect of wheat seed extract is greater than effect of the triticale seed extract on the enzyme activity. Lowest concentration of wheat and triticale seed extracts (0.106 mg protein/mL) inhibited 39% and 18% of the enzyme activity, respectively (Figure 4). Thus, wheat seed extraction concentrations of 1.7 mg protein/mL, 0.85 mg protein/mL, 0.425 mg protein/mL, 0.212 mg protein/mL and 0.106 mg protein/mL caused 82.0%, 76.24%, 74.21%, 55.64%, 39.52% enzyme inhibition, respectively. Similar concentrations of Triticale seed extraction caused 75.5%, 65.64%, 51.46%, 46.42% and 17.29% enzyme inhibition, respectively. Therefore, the highest concentration of the two seed extracts including wheat and triticale inhibited 82% and 75% enzyme activity, respectively showing potency of the wheat seed extract against this insect alpha amylase activity. 

Figure 4 Inhibition of Ectomyelois ceratoniae α-amylase by wheat and triticale seed extract

2.3 Effects of the seed extracts on protease activity
Changes in the activity of protease enzymes in the presence of seed extracts showed a dose dependent manner (Figure 5) although there were not significant changes in the enzyme activity between the two seed extracts. Concentrations of 1.7 mg protein/mL, 0.85 mg protein/mL, 0.425 mg protein/mL, 0.212 mg protein/mL and 0.106 mg protein/mL triticale seed extract inhibited the insect protease activity 56.86%, 26.14%, 20.26%, 7.18% and 2.61%, respectively (Figure 5). Wheat seed extraction at concentrations of 1.7 mg protein/mL, 0.85 mg protein/mL, 0.425 mg protein/mL, 0.212 mg protein/mL and 0.106 mg protein/mL inhibited the insect gut protease 61.56%, 23.52%, 21.45%, 17.64% and 15.22%, respectively. 

Figure 5 The effect of wheat and triticale seed extractions on the protease activity of the carob moth gut

At the lowest concentration used (0.106 mg protein/mL) the enzyme activity was inhibited 2.6% and 15.0% for triticale and wheat seed extracts, respectively. However, at the highest concentration of the triticale and wheat seed extracts (1.7 mg protein/mL) percentage of inhibition was 56.0% and 61.0%, respectively.

2.4 In gel amylase and protease assays
When amylase was assayed in the gel, two bands were seen in all three parts. However, the middle part of the midgut bands were clearly bigger than those of the others indicating that the enzyme isoforms are more active in the middle parts (Figure 2B). Relative mobility of A1 band was 0.25 and A2 band was 0.43.

In the protease gel, two protease bands were seen in the mid, anterior and posterior midgut and similar to the amylase bands, protease bands of the mid part were sharper than the anterior and posterior midgut showing the higher activity of these proteases in the middle parts (Figure 3B). Relative mobility of the P1 and P2 were 0.52 and 0.6, respectively.

2.5 The effect of wheat and triticale seed extracts on α-amylase in gel assays
The effect of wheat and triticale seed extract on α-amylase activity in the gel was similar to the effect of these plants extracts on spectrophotometric enzyme assays i.e. with increasing the seed extraction the amount of the enzyme inhibition increased, too (Figure 6). As shown in figure 5 in first lane two bands seen but in the other lanes where triticale seed extracts were used second amylase band (A2) was eliminated and the first amylase band (A1) intensity was decreased. The same pattern of inhibition was seen when wheat seed extract was used in enzyme assay (Figure 7). In control (a) no inhibition was observed but in the second lane (b) which the greatest amount of the wheat seed extract was used (1.7 mg protein/mL) the least intensity of the band is seen when compared with last lane (f) which the least amount of the wheat seed extracts (0.16 mg protein/mL) was tested.

Figure 6 The effect of the triticale seed extracts on the protease activity of the carob moth gut

Figure 7 The effect of the wheat seed extracts on the α-amylase activity of the carob moth gut

 
2.6 The effect of wheat and triticale seed extracts on protease activity in gel assay
Effect of the wheat seed extract on protease activity of the gut was dose dependant (Figure 8). When high dose of the extract (1.7 mg protein/mL) was used almost all the two protease bands were eliminated and as the wheat seed extract dose reduced the intensity of the two bands increased (Figure 8). The same pattern of the protease inhibition observed when triticale seed extract was used (Figure 9). Triticale seed extract affected the insect protease activity in a dose-dependent manner, as with wheat seed extract. So, at the highest dose used the P2 band was completely disappeared and trace amount of the P1 band left. The effect of the wheat seed extract on the insect gut protease was more than triticale seed extract since the high dose (1.7 mg protein/mL) of the wheat and triticale seed extracts inhibited the protease activity more than 70% and less than 60%, respectively.

Figure 8 The effect of the triticale seed extracts on the protease activity of the carob moth gut

Figure 9 The effect of the wheat seed extracts on the protease activity of the carob moth gut

 
3 Discussion
In this study it was found that there are two α-amylase (A1 and A2) isoenzymes and two protease found in the carob moth digestive system. Presence of different isoenzymes in insects shows its importance in insect digestion of carbohydrate to guarantee effective digestion (Mehrabadi et al., 2012). There are many reports that some species have more than one isoenzyme in their digestive system showing their important role in insect metabolism (Mehrabadi et al., 2010). Also, the presence of a number of α-amylases isoenzymes is a strategy to escape plant secondary metabolites (Silva et al., 1999). Production of more than one α-amylase also was detected in the other insect species including Sitophilus zeamais, Callosobruchus maculatus, Zabrotes subfasciatus, Acanthoscelides obtectus, and Eurygaster integriceps (Silva et al., 1999; Franco et al., 2005; Bandani et al., 2009).

Some insects such as carob moth are somehow dependent on α-amylases for their survival so these enzymes could be a good target for insect control through digestive enzyme inhibitors (Franco et al., 2002; Svensson et al., 2003; Sivakumar et al., 2006). Moreover, there are many repots that these plant defense molecules are abundant in cereals and legumes, which play an important role in plant defenses against pests and pathogens (Franco et al., 2002; Sivakumar et al., 2006). So, in this study the effect of two cereal species, triticale and wheat, seeds extracts were tested against carob moth digestive α-amylase and proteases enzymes. It was found that wheat seed extract had more inhibitory effect on the carob moth enzymes (α-amylases and proteases) than triticale seed extract. However, the effect of the both wheat and triticale seed extract on α-amylases were more than proteases. Also, wheat seed extract had more inhibitory effect on α-amylases than triticale seed extract i.e. wheat seed extract inhibited the insect alpha amylase 82% and triticale seed extract inhibited the insect alpha amylase 75%. Mehrabadi et al. (2010) showed that triticale seed extract inhibited the Sunn pest (Eurygaster integriceps) α-amylase activity about 80%. Also, in this study it was found that wheat and triticale seed extracts inhibited general protease activity of the carob moth 62% and 57%, respectively. So, interestingly, in this study it was found that wheat seed extract have more inhibitory activity on both alpha amylase and protease of the carob moth than triticlae seed extract. These data shows that proteinaceous extract of different plant species shows different specificity toward different insect species which is an important step in developing molecules for production of insect resistant transgenic plants (Valencia et al., 2000; Bandani et al., 2009).

Overall, these data are in agreement with the finding of the other researcher (Valencia et al., 2000) which shown that a dose dependent inhibition of the Amaranthus cruentus against α-maylase of coffee berry borer, Hypothenemus hampei. Mehrabadi et al. (2012) showed that triticale seed extract affect E. integriceps salivary enzyme as well as midgut enzymes. Also, they found that triticale seed extract is resistant to the insect enzymes so they concluded that thiticale seed extract has potential to be investigated further in order to determine its potential application in integrated pest management program (IPM).

Gel assays using native gel confirmed results obtained from spectrophotometric assay showing the greatest effect of the wheat seed extract on the carob α-amylase than protease. Also, these results confirmed that the effect of seed extracts on the digestive enzyme was in a dose dependant manner because as dose is increased either enzyme band/s was/were disappeared or intensity of the enzyme band/s was/were decreased.

In conclusion, different plant species produces different proteins with different specificity toward insect digestive enzymes that these proteins could be used in plant protection strategies. Specificity of inhibitors is an important issue since the introduced inhibitors must not adversely affect the plant’s own enzymes as well as non target organisms including mammals’ enzymes.

4 Materials and Methods
4.1 Insects rearing
A population of E. ceratoniae was collected from Chandab Region, Varamin, Tehran province, Iran. The larvae were reared on artificial diet under laboratory conditions at (29.6 ± 5)℃, with a 16:8 h photoperiod and (75 ± 5)% RH as described by Norouzi et al. (2008) with some modifications.

4.2 Sample preparation
Dissection of the insect gut and sample preparation was done based on Kazzazi et al. (2005). Briefly, fourth larval instar gut was removed by dissection under a light microscope. Then, the removed gut placed in pre-cooled homogenizer. Homogenization was done in ice cold distilled water and samples were centrifuged at 15 000 g for 15 min at 4℃, the supernatant was removed and stored at −20℃ for subsequent use.

4.3 α-amylase and Protease assays
α-amylase activity of gut was assayed by the dinitrosalicilic acid (DNS) procedure (Bernfeld, 1955), using 1% soluble starch solution as substrate as described by Bandani et al. (2009). One unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35℃. A standard curve of absorbance against amount of maltose released was constructed to enable calculation of the amount of maltose released during α-amylase assay. A blank without substrate but with α-amylase extract and a control containing no α-amylase extract with substrate were run simultaneously with reaction mixture. All assays were performed in triplicates and with three replications.

General protease assay was done according to the methods of Elpidina et al. (2001) and Gatehouse et al. (1999), with slight modification. Briefly, 10 µL enzyme extract and 50 µL substrate solution (Azocasein 2%) were mixed with 40 µL 20mM Glycine-NaOH buffer (pH 10). After 60 min incubation, 100 µL 30% trichloroacetic acid (TCA) was added to the reaction mixture, and kept it at 4℃ for 30 min, followed by centrifugation at 15 000 g for 15 min to precipitate non hydrolysis substrate. 100 µL 1 M NaOH was added to 100 µL supernatant and the absorbance at 405 nm was measured.

4.4 Seed protein extraction
Wheat and triticale seed proteins were extracted according to Baker (1987) and Melo et al. (1999). Seed was powdered thoroughly, and then 30 grams of powdered seeds from each plant separately was mixed with a solution of 0.1 M NaCl and stirred for 3 h, followed by centrifugation at 8 000 g for 30 min. The pellet was discarded, and the supernatant was placed at 70℃ for 20 min to inactivate enzymes within the seeds. Seed protein was extracted using a saturation of 70% ammonium sulfate followed by centrifugation at 8 000 g for 30 min at 4℃. The pellet containing the highest fraction of amylase inhibitors was dissolved in ice-cold sodium phosphate buffer (0.02 M and pH 7.0) and was dialyzed against the same buffer for 20 h. This dialyzed solution was used as inhibitors in enzymatic assay tests.

4.5 The effect of inhibitor on α-amylase activity
The effects of the seed extracts on the amylase activities were determined as described by Mehrabadi et al. (2010). Enzyme extract was pre-incubated with Triticale and three cultivars of wheat seed extracts for 30 min at 35℃ followed by determination of the enzyme activity as described before using dinitrosalicilic acid (DNS) procedures. Appropriate blanks were included in the experiments as well. The inhibition percentage (%I) was calculated as follow:

%I=100×[(A540 control−A540 Exp)/A540 control]

4.6 The effect of inhibitor on general protease activity
The effects of the seed extracts on the general protease activities were determined as described by Saadati et al. (2011). Enzyme was pre-incubated with triticale and three cultivars of wheat seed extracts for 30 min at 35℃ followed by determination of the enzyme activity as describe before. The inhibition percentage was calculated as described for amylase.

4.7 In gel assay
Amylolytic activity in the gel was detected using procedures described by Meharbadi et al. (2011). Briefly, PAGE was performed in 10% (w/v) gel for separating gel and 5% for stacking gel with 0.05% SDS. Electrophoresis was conducted at a voltage of 120V until the blue dye reached the bottom of the gel. The gel was rinsed with distilled water and washed by 1% (v/v) Triton X-100 buffer for 15 min. Then, the gel was incubated in Glycine-NaOH buffer (pH 9) containing 1% starch solution, 2 mM CaCl₂ and 10 mM NaCl for 1.5 h. Finally, the gel treated with a solution of 1.3% I₂ and 3% KI to stop the reaction and to stain the un-reacted starch background. Zones of α-amylase activities appeared at light band against dark background.

Electrophoretic detection of proteolytic enzymes was done basically according to the procedures described by Laemmli (1970) and Walker et al. (1998). PAGE was performed in 10% (w/v) gel for separating that co-polymerized with 0.1% gelatin and 4% for stacking gel with 0.05% SDS. Electrophoresis was conducted at 4℃ until the blue dye reached the bottom of the gel. Then, the gel was rinsed with distilled water and washed by 2.5% (v/v) Triton X-100 buffer for 60 min followed by incubation in Glycine-NaOH buffer (pH 10) for about 6 hour. Finally, the gel was stained as described by Saadati et al. (2011).

4.8 Protein determination
Protein concentration was measured according to the method of Bradford (1976), using bovine serum albumin as a standard.

4.9 Statistical analysis
Data was analysed based on a completely randomized design using SAS software. Mean comparison was done using Duncan’s test.

Authors’ Contributions
Ehsan is a student and carried out the experiments and drafted the manuscript. Ali is Ehsan’s supervisor and participated in the design of the study, helped in statistical analysis and correction of the manuscript. Ali also paid from his grant for the project expenditure. Both authors read and approved the final manuscript.

Acknowledgements
This work was funded by a grant from University of Tehran.

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