Layout 1 [Journal of Entomological and Acarological Research 2014; 46:1868] [page 35] Abstract The digestive proteolytic profile of Apodiphus amygdali was deter- mined by using several substrates and specific inhibitors. Analysis of optimal pH and temperature showed the highest enzymatic activity at the pH range of 6-7 and temperature of 40°C when azocasein was used as a substrate. By using a negative control, the presence of several spe- cific proteases were determined including tryspin-like, chymotrypsin- like, elastase, cathepsin B, cathepsin L, amino- and carboxypeptidases in the midgut content of A. amygdali, with the highest and the lowest activities of cathepsin L and carboxypeptidase, respectively. pH dependency of specific proteases revealed optimal pHs of 9, 8 and 9 for trypsin-, chymotrypsin-like, 6 for cathepsins and 5-6 for carboxy- and aminopeptidases, respectively. Specific inhibitors, including phenyl- methylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, N- tosyl-L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinyl- leucylamido-(4-guanidino)-butane, phenanthroline and ethylendi- amidetetraacetic acid, significantly decreased proteolytic activity, indi- cating the presence of different proteases in the midgut of A. amygdali. Extracted inhibitors from the midgut demonstrated significant inhibi- tion of specific proteolytic activities of A. amygdali except for cathep- sin B and aminopeptidase. The results indicated that determination of digestive proteolytic activity could be helpful to clarify digestion process in insects. Moreover, understanding the nature of digestive proteases might be used to develop several inhibitors for providing resistant crop varieties against pests. Introduction Apodiphus amygdali Germar (Hemiptera: Pentatomidae) is a polyphagous and univoltine hemipteran distributed throughout Europe and the Middle East (Schuh & Slater 1995). It feeds on several fruit trees, especially plum, apricot, apple, olive, pear and pistachio as well as non-fruit trees like poplar, pine, plane-tree, elm and willow bark (Muhammed & Al-Iraqi, 2010). Nymphs and adults feed on leaves, fruits and flowers by injecting their salivary secretions into the plant tissues to liquefy nutrient materials. Feeding on stems and fruits caus- es host weakness and attracts other insects to feed on host plants. Feeding on fruits is the primary damage and causes complete degrada- tion and yield loss (Schuh & Slater, 1995). Digestive proteases are one of the crucial enzymes in the alimenta- ry canals of insects since proteins are the key nutrients for growth, development and reproduction (Nation, 2008). The enzymes are clas- sified based on their activity against protein molecules. In fact, pro- teases that attack internal bonds are called endopeptidases (proteinas- es) but exopeptidases refers to proteases separating amino acids from N- and C-terminal of proteins, known as aminopeptidases (EC 3.4.11.2) and carboxypeptidases (EC 3.4.11.7) (Terra & Ferreira, 2012). Endopeptidases are divided into different subclasses based on their pH dependency and catalytic sites. Serine proteases, including trypsin- (EC 3.4.21.4), chymotrypsin-like (EC 3.4.21.1) and elastase (EC 3.4.21.36), are active in alkaline pH and they have serine, histi- dine, and aspartic acid residues in their catalytic sites (Terra & Ferreira, 2012). Meanwhile, these enzymes have different structural features that are associated with their different substrate specificities (Terra & Ferreira, 2012). Cysteine proteases are active at acidic pH, including cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15). Cathepsin L (EC 3.4.22.15) is a true endopeptidase that preferentially cleaves peptide bonds in P2 against the hydrophobic amino acid residues, but cathepsin B prefers arginine at the same position (Barrett et al., 1998). Digestion of proteins is initiated by endopeptidases; due to the activity of endopeptidases, oligopeptidases are attacked from the N- and C-terminal by amino- and carboxypeptidases, resulting in dipep- tides that are hydrolyzed by dipeptidases (Terra & Ferreira, 2012). Since synthetic chemicals cause severe negative effects on the envi- ronment, non-target organisms and induce resistance in pests, numer- ous studies have been conducted on the use of biocontrol agents and digestive enzyme inhibitors against agricultural pests. Enzymatic inhibitors disrupt digestion process of insects and nutrient absorption, thereby decreasing reproduction and therefore pest populations. Protease inhibitors are small proteins that are present in up to 100 plant Correspondence: Arash Zibaee, Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, 416351314, Iran. Tel.: +98.0131.6690274 - Fax: +98.0131.6690281. E-mail: arash.zibaee@gmx.com ; arash.zibaee@guilan.ac.ir Key words: Apodiphus amygdali, digestive protease, inhibitor. Acknowledgements: the authors want to thank Dr. Ali Sarafrazi from Iranian Institute of Plant Protection for his contribution in identification of collect- ed insects. Received for publication: 7 August 2013. Revision received: 19 November 2013. Accepted for publication: 19 November 2013. ©Copyright S. Ramzi and A. Zibaee, 2014 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2014; 46:1868 doi:10.4081/jear.2014.1868 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Journal of Entomological and Acarological Research 2012; volume 44:eJournal of Entomological and Acarological Research 2014; volume 46:1868 Digestive proteolytic activity in Apodiphus amygdali Germar (Hemiptera: Pentatomidae): effect of endogenous inhibitors S. Ramzi, A. Zibaee Department of Plant Protection, University of Guilan, Rasht, Iran No n- co mm er cia l u se on ly [page 36] [Journal of Entomological and Acarological Research 2014; 46:1868] species and they are effective on serine and cysteine proteases (Nation, 2008). Although this approach may be suitable for decreasing economic losses caused by pests with the least environmental disruption, compre- hensive experiments are required to achieve the final goal. Determination of proteolytic profiles and their properties are the first and fundamental step in this approach. The objectives of the current study were to determine digestive proteolytic profiles in A. amygdali, the effect of synthetic inhibitors, and to extract endogenous inhibitors. Materials and methods Insect rearing Fifth nymphal instars of A. amygdali were collected from elm trees in Shiraz (Fars province, Iran) and transferred to the laboratory. The nymphs were reared on elm leaves at 28±1°C, 70% relative humidity and a 16L:8D of photoperiod. When adults emerged, they were allowed to feed on leaves for 48 h prior to being randomly selected for biochem- ical analysis. Sample preparation Based on the method of Cohen (1993), adults of A. amygdali were randomly selected and their midgut removed by dissection in iced saline solution (NaCl, 10 mM, 1.06404.1000, Merck-Chemicals, Darmstadt, Germany). Integument and unneeded organs were removed and the midgut was gently separated and rinsed in 1 mL of iced distilled water. To obtain appropriate samples, five midguts were placed in one Eppendorff tube (Eppendorff, Hamburg, Germany) containing 1 mL of distilled water. Tissues were ground using a homogenizer and cen- trifuged at 13,000 rpm for 20 min at 4°C (Daika, Rika Kugyo Co., Tokyo, Japan). The supernatant was carefully removed, transferred to new tubes and stored at –20°C for no more than one week until use in the experiments. General proteolytic assay Azocasein (2%; A2765; Sigma-Aldrich Co., St. Louis, MO, USA) was used to find the general proteolytic activity in the midgut of A. amygdali adults. General proteolytic activity was measured by using azocasein 2% based on the method of Elpidina et al. (2001). The reaction mixture consisted of 50 mL of appropriate buffer solutions (Universal buffer, 0.02 M, containing succinate, glycine and 2-morpholinoethanesulfonic acid; pH range 3-12; Frugoni, 1957), 20 mL azocasein and 20 mL of the enzyme solution. After incubation at 37°C for 60 min, proteolysis was stopped by adding 100 mL of 30% trichloroacetic acid (TCA). Precipitation was achieved by cooling at 4°C for 10 min; it was then centrifuged at 13,000 rpm for another 10 min. An equal volume of 2 M NaOH was added to the supernatant and the absorbance was recorded at 450 nm (Awareness Technology Inc., Palm City, FL, USA). A blank solution consisted of all the above-mentioned portions except the for enzyme solution. Optimal pH and temperature (°C) for general proteolytic activity The reaction mixture was the same as described above, but different pH ranges of universal buffer (from 3 to 12) and temperature (from 20 to 70°C) were used to determine optimal pH and temperature. For the optimal pH assays, 40 mL of buffer solution (at different pHs) was incu- bated with azocasein as a substrate for 10 min at 30°C (Terra & Ferreira, 2012). Then, 20 mL of sample was added and the experiment continued as above. For the optimal temperature assays, 40 mL of buffer solution (at the optimal pH found) was incubated with azocasein as a substrate for 10 min at different temperature regimes from 20 to 70°C. After adding 20 mL of the sample, the reaction mixture was incubated at each given temperature for 60 min. Other steps were conducted as described above. Specific protease assays Serine proteases Trypsin-, chymotrypsin- and elastase-like activities (as three sub- classes of serine proteases) were assayed using a 1 mM concentra- tion of BApNA (Nabenzoyl-L-arginine-p-nitroanilide, Sigma-Aldrich, 19362), 1 mM SAAPPpNA (N-succinyl-alanine-alanine-proline-pheny- lalanine-p-nitroanilide, Sigma-Adrich, S7388) and 1 mM SAAApNA (N-succinyl-alanine-alanine-alanine-p-nitroanilide, Sigma-Aldrich, S4760) as substrates, respectively. The reaction mixture consisted of 35 mL of universal buffer (pH 8, as the recommended pH for serines in the literature), 5 mL of each substrate, and 5 mL of enzyme solu- tion. The reaction mixture was incubated at 30°C for a period of 0-10 min before adding 30% TCA to terminate the reaction. The absorbance of the resulting mixture was then measured spectropho- tometrically at 405 nm by p-nitroaniline release. To prove the specif- ic proteolytic activity, a negative control for each substrate was pro- vided separately containing all the above components except for enzyme pre-boiled at 100°C for 30 min (Oppert et al., 1997). Cysteine proteases Cathepsins B and L activities (as three subclasses of cysteine pro- teases) were assayed using a 1 mM concentration of Z-Ala-Arg-Arg 4- methoxy-b-naphtylamide acetate (Sigma-Aldrich, C8536) and N- Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (Sigma-Aldrich, B2133) as substrates, respectively. The reaction mixture consisted of 35 mL of universal buffer (pH 5 as the recommended pH for cysteines in the literature), 5 mL of each substrate and 5 mL of enzyme solution. The reaction mixture was incubated at 30°C for a period from 0-10 min before adding 30% TCA to terminate the reaction and read at 405 nm. To prove the specific proteolytic activity, a negative control for each substrate was provided separately containing all the above components except for enzyme pre-boiled at 100°C for 30 min (Oppert et al., 1997). Exopeptidases The activities of two exopeptidases were obtained by using Hippuryl- L-Arginine (Sigma-Aldrich, H2508) and Hippuryl-L-Phenilalanine (Sigma-Aldrich, H6875) for amino- and carboxypeptidases in the midgut of A. spinidens. The reaction mixture consisted of 35 mL of uni- versal buffer (pH 7 as the recommended pH in the literature for exopeptidases), 5 mL of each substrate and 5 mL of enzyme solution. The reaction mixture was incubated at 30°C for a period from 0-10 min before adding 30% TCA to terminate the reaction and read at 340 nm. To prove the specific proteolytic activity, a negative control for each substrate was provided separately containing all the above components except for enzyme pre-boiled at 100°C for 30 min (Oppert et al., 1997). Optimal pH determination of specific proteases Universal buffer was used to find the optimal pH of serine and cys- teine proteases in addition to exopeptidases by using the specific sub- strates described. The reaction mixture and experiment procedure were the same as above. Effect of specific inhibitors on proteolytic activity The following compounds were used to find any alteration of the pro- teolytic activity in the midgut of A. spinidens: phenylmethylsulfonyl flu- oride (PMSF) (Sigma-Aldrich, P7626); trypsin inhibitor, Na-p-tosyl-L- lysine chloromethyl ketone (TLCK) (Sigma-Aldrich, T5012); chy- motrypsin inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone Article No n- co mm er cia l u se on ly (TPCK) (Sigma-Aldrich, T7254); cysteine protease inhibitor, L-trans- epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64) (Sigma- aldrich, E3132); cystatin (Sigma-Aldrich, C8917), metalloprotease inhibitors including phenanthroline (Sigma-Aldrich, 131377) and eth- ylendiamidetetraacetic acid (EDTA) (Merck-Chemicals). Extraction of endogenous inhibitors Endogenous inhibitors from the midgut of A. amygdali were extract- ed by the method of Lwalaba et al. (2010) with slight modifications. Midguts of adults (N=10) were incubated in 500 mL of low glucose Ringer’s solution (121.5 mM NaCl; 10 mM KCl; 2.1 mM NaH2PO4; 10 mM NaHCO3; 0.7 mM MgCl2; 2.2 mM CaCl2; pH=6.8 and 0.01 g glucose) for 30 min at 30°C. The tissues were then ground using a glass homog- enizer. These samples taken from different species were heated to 90°C for 30 min. Samples were centrifuged at 13,000 rpm for 20 min at 4°C. The obtained supernatants were used as the source of inhibitors. Effect of endogenous inhibitors on specific proteolytic activity The reaction mixture contained 50 mL of universal buffer, 20 mL of each substrate and 20 mL of endogenous inhibitor. After 5 min of incubation, 10 mL of the enzyme was added and proteolytic activity was read at 405 nm after 10 min. A blank contained all components except the enzyme. Protein assay Protein concentration was measured according to the method of Lowry et al. (1951). In this method, peptide bonds are oxidized by Folin- Ciocalteu reagent. Briefly, 20 mL of sample was added to 100 mL of reagent, which was incubated for 30 min prior to reading the absorbance at 545 nm (Recommended by Ziest Chem. Co., Tehran, Iran). Statistical analysis The experimental design was based on a completely randomized design and Tukey’s test was used to compare treatment means. Statistical differences were considered at P≤0.05. Results from the syn- thetic inhibitors trials were analyzed using POLO-PC software to calcu- late inhibitory concentration (IC) values. Results Optimal pH and temperature of general proteolytic activity Determination of the optimal pH for general proteolytic activity resulted in two peaks in the acid and alkaline ranges. The larger peak was observed at the pH range of 6-7 and the smaller peak was found at the pH range of 9-10 (Figure 1A; F=40.99, PrF: 0.0001). Discussion and conclusions A critical role of proteases could be expected in the midgut of A. amygdali, as this is a polyphagous hemipteran that feeds on different trees. In the current study, several proteases were found in the midgut, although cysteine proteases, mainly cathepsin L, showed the highest activity among the proteases assayed. Results of other studies partially correspond with our findings. Stamopoulos et al. (1993) and Bell et al. (2005) demonstrated trace activities of trypsin and chymotrypsin in the gut of Podisus maculiventris Say (Hemiptera: Pentatomidae). Silva & Terra (1994) reported the presence of cysteine proteases in the midgut of Dysdercus peruvianus Guérin-Méneville (Hemiptera: Pyrrhocoridae). Pascual-Ruiz et al. (2009) reported serine, cysteine, aminopeptidase and carboxypeptidase as the main proteases involved in protein diges- tion of P. maculiventris, although their activities can differ due to vari- ability of prey. Zhu et al. (2003) and Wright et al. (2006) found high activities of cysteine and serine proteases in the midgut of Lygus line- olaris and L. Hesperus Knight (Hemiptera: Miridae). Bigham & Hosseininaveh (2010) found the presence of cathepsins B and L in the Article Figure 3. Optimal pH for serine proteases (A-C) in the midgut of A. amygdali. Letters show statistical differences among values (Tukey’s test, P≤0.05). Figure 4. Optimal pH for cysteine proteases (A and B) in the midgut of A. amygdali. Letters show statistical differences among values (Tukey’s test, P≤0.05). No n- co mm er cia l u se on ly midgut of Brachynema germari Kolenati (Hemiptera: Pentatomidae). Fialho et al. (2012) obtained cathepsin L and aminopeptidase as the major proteases from the midgut of Podisus nigrispinus Dallas (Hemiptera: Pentatomidae). Sorkhabi-Abdolmaleki et al. (2013) report- ed both serine and cysteine proteases through the significant role of cysteines in the midgut content of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae). The optimal pH for general proteolytic activity in the midgut of A. amygdali was represented as two peaks at both acidic and alkaline pHs, indicating the presence of all the determined proteases (see above). Zhu et al. (2003) found optimal proteolytic activity of Lygus lineolaris Knight (Hemiptera: Miridae) at pH 4.25 and 8.5. Wright et al. (2006) found acidic and neutral pHs for caseinolytic activity in L. lineolarius that might be attributed to cysteine and serine proteases. Bigham & Hosseininaveh (2010) observed the presence of general protease activity by acidic and alkaline optima in the midgut extract of B. germari using hemoglobin as a substrate. Sorkhabi-Abdolmaleki et al. (2013) found an alkaline pH for general proteolytic activity, serine proteases and two exopeptidases with another peak at pH 6, as with the cysteine proteases. The optimal pH of specific proteases corresponds with the expected values, as noted in the literature (Terra & Ferreira, 2012). A pH peak was observed for all spe- cific proteases except for cathepsin L, for which the enzyme was active at a pH range of 4-6. This could be attributed to the presence of several isozymes and higher activity of cathepsin L. Variations in optimal pH of general proteolytic activity in insects could be attributed to feeding habits as either monophagous or polyphagous. Since polyphagous insects feed on various hosts, the presence of different proteases and the difference in general protease pH could be attributed to host materials or secondary metabolites of plants. An optimal temperature of 40°C was found for general proteolytic activity, although there was slightly lower but not statistically different activity at 35°C. Generally, optimal temperature of an enzyme in in vitro assays reflects the temperature of the environment where the organism feeds on its hosts. Increasing temperature can disrupt hydro- gen bonds in the three-dimensional structure of the molecule, leading to denaturation of the protein (Zeng et al., 2000). At the same time, bio- logical reactions occur faster with increasing temperature, up to the point of enzyme denaturation, where the enzymatic activity and the rate of the reaction decrease sharply (Zeng et al., 2000). Swart et al. (2006) found 37°C as the optimal temperature for proteolytic activity in the salivary secretion of two belostomatid bugs. Sorkhabi-Abdolmaleki [Journal of Entomological and Acarological Research 2014; 46:1868] [page 39] Article Figure 6. Effect of specific inhibitors on general proteolytic activ- ity in the midgut of A. amygdali. A) Specific inhibitors of serine proteases; B) Specific inhibitors of cysteine proteases; C) Specific inhibitors of metalloproteinase. Letters show statistical differ- ences among values (Tukey’s test, P≤0.05). PMSF, phenylmethyl- sulfonyl fluoride; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; E- 64, L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane; EDTA, ethylendiamidetetraacetic acid. Figure 5. Optimal pH for exopeptidases (A and B) in the midgut of A. amygdali. Letters show statistical differences among values (Tukey’s test, P≤0.05). No n- co mm er cia l u se on ly [page 40] [Journal of Entomological and Acarological Research 2014; 46:1868] & Zibaee (2013) reported an optimal temperature of 25°C for proteolyt- ic activity in the midgut of A. spinidens that corresponded with labora- tory conditions of mass-rearing. Our results demonstrated inhibitory effects of PMSF, TLCK and TPCK on the proteolytic activity in the midgut of A. amygdali. PMSF showed the highest inhibition of enzymatic activity with the lowest IC50 value (0.574 mM), in comparison with TLCK (7.13) and TPCK (5.19). These results corresponded with previous results on the presence of serine proteinas- es, especially chymotryptic activities, in the midgut of A. amygdali, which have been supported by using a negative control and specific substrates. Zhu et al. (2003) found serine proteases as the major enzymes in the gut of L. lineolaris by TLCK (Zhu et al., 2003). Bigham & Hosseininaveh (2010) and Sorkhabi et al. (2013) found significant inhibition of diges- tive proteases by PMSF, TLCK and TPCK in B. germary and A. spinidens. Presence of cysteine proteases was confirmed by using specific sub- strates, cystatin, E-64 and DTT, although the experiment with a negative control showed cathepsin L as the major protease in the midgut of A. amygdali. Houseman and Dowe (1983) found cathepsin B and L activi- ties in the posterior midgut of Rodnius prolixus L. (Hemiptera: Reduviidae). Overney et al. (1997) reported cysteines as the major pro- teases in the midgut of Perillus bioculatus Fabricius (Hemiptera: Pentatomidae), showing inhibition of 90% by E-64. Proteolytic activity in the midgut of L. lineolaris was significantly decreased with E-64 (Wright et al., 2006). Bigham & Hosseininaveh (2010) found negative effects of E-64 on the proteolytic activity of A. spinidens. Sorkhabi-Abdolmaleki et al. (2013) observed that E-64 and cystatin decreased proteolytic activity in the midgut of A. spinidens. EDTA as general chelating agent and phenanthroline as metallopro- teinase inhibitor significantly decreased proteolytic activity in the midgut of A. amygdali, indicating the presence of metal ions in the active sites of aminopeptidase. Aminopeptidases are divided into aminopeptidase A and N based on the presence of Zn2+ and Mn2+ in their active sites (Terra & Ferreira, 2012). There is little knowledge on the characterization of aminopeptidases’ active site. Ferreira & Terra (1986) employed multiple inhibition analysis to find the possible role of EDTA on inactivation of aminopeptidase in Rhynchosciara ameri- cana Wideman (Diptera: Sciaridae) larvae. The authors found two sub- sites in the active center of the enzyme: a hydrophobic subsite to which isoamyl alcohol binds, exposing the metal ion, and a polar subsite, to which hydroxylamine binds (Ferreira & Terra, 1986). Exposure of the metal ion after isoamyl alcohol binding may be analogous to the situa- tion when a part of the substrate occupies the hydrophobic subsite to cause conformational changes associated with the catalytic step (Ferreira & Terra, 1986). Cristofoletti & Terra (2000) found that the catalysis property of aminopeptidase in Tenebrio molitor L. (Coleoptera: Tenebrionidae) depends on a metal ion, a carboxylate and a protonated imidazole group. Detailed experiments revealed that the enzyme is a zinc metallopeptidase like mammalian aminopeptidase N, but it differs in some details (Cristofoletti & Terra, 2000). Extraction of endogenous inhibitors from the midgut of A. amygdali revealed diverse effects on specific proteolytic activities. In serine pro- teases, inhibition was more significant in the case of trypsin and elas- tase than chymotrypsin. In the case of cysteine and exopeptidases, cathepsin B and aminopeptidase were not affected by the endogenous inhibitor. Endogenous inhibitors may have several functions, such as protection of a-amylase from proteolytic activity (Stein & Fischer, 1958), attacking fungi and bacteria in gut (Taranushenko et al., 2009) and pro- tection of epithelial cells from digestive enzymes when food is not pres- ent in the midgut (Lwalaba et al., 2010). Engelmann & Geraert (1980) reported extraction of the midgut lumen content in Leucophaea madera Fabricius (Blattodea: Oxyhaloidae) as an endogenous inhibitor. Lwalaba et al. (2010) extracted an endogenous inhibitor from the midgut of Spodoptera frugiperda Hubner (Lepidoptera: Noctuidae) larvae. It was found that the ability of the endogenous inhibitor to decrease trypsin activity was similar to the results from ingestion of the exogenous inhibitor SBTI. Zibaee et al. (2012) and Sorkhabi-Abdolmaleki et al. (2013) extracted endogenous inhibitors from Chilo suppressalis Walker (Lepidoptera: Crambidae), Naranga aenescens Moore (Lepidoptera: Noctuidae), Pieris brassicae L. (Lepidoptera: Pieridae), Hyphantria cunea Drury (Lepidoptera: Arctiidae) and Ephestia kuhniella Zeller (Lepidoptera: Pyralidae). The authors found different effects of inhibitors on salivary and midgut proteases of A. spinidens (Zibaee et al., 2012; Sorkhabi-Abdolmaleki & Zibaee, 2013). Specifically, extracted inhibitors from C. suppressalis, N. aenescens and E. kuehniella signifi- Article Table 1. Determination of inhibitory concentrations (mM) of specific inhibitors on proteolytic activity in the midgut of A. amygdali. Compound IC10 IC30 IC50 Slope±SF χ2 PMSF 0.045e 0.574f 0.574e 1.16±0.27 0.568 TLCK 3.57b 5.38a 7.13ab 4.27±0.40 25.27 TPCK 3.29b 4.65b 5.91b 5.04±0.39 37.46 Cystatin 1.99c 3.18c 4.41c 3.71±0.29 17.44 E-64 1.39cd 2.75cd 4.41c 2.55±0.25 0.293 DTT 0.59d 1.56e 3.07d 1.79±0.24 39.21 Phenanthroline 3.44b 5.43a 7.46a 3.81±0.38 2.79 EDTA 4.33a 5.80a 7.10ab 5.95±0.52 21.49 IC, inhibitory concentrations; PMSF, phenylmethylsulfonyl fluoride; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; E-64, L-trans-epoxysuccinyl-leucylamido-(4- guanidino)-butane; DDT, dithiothreitol; EDTA, ethylendiamidetetraacetic acid. a,b,c,d,e,fLetters show statistical differences among values (Tukey’s test, P≤0.05). Table 2. Effect of endogenous inhibitor on specific proteolytic activity in the midgut of A. amygdali. Tryspin Chymotrypsin Elastase Cathepsin B Cathepsin L Aminopetidase Carboxypeptidase Control 1.59±0.02* 0.82±0.01* 1.10±0.11* 0.55±0.05 1.04±0.024* 0.094±0.009 0.16±0.003* Treatment 0.15±0.025 0.23±0.06 0.046±0.003 0.65±0.05 0.015±0.012 0.17±0.001* 0.024±0.001 *Asterisks show statistical differences among values (Student’s t-test, P≤0.05). No n- co mm er cia l u se on ly cantly decreased trypsin and chymotrypsin activities. All extracted inhibitors significantly decreased elastase and cathepsin activities of A. spinidens except for C. suppressslis (Sorkhabi-Abdolmaleki & Zibaee, 2013). Extracted inhibitors from all species significantly decreased cathepsins B and L activities, but this decrease was not significantly dif- ferent in the case of C. suppressalis. In the case of exopeptidases, endogenous inhibitors from E. kuehniella and H. cunea on aminopepti- dase and all inhibitors on carboxypeptidase significantly decreased enzy- matic activities (Sorkhabi-Abdolmaleki & Zibaee, 2013). In conclusion, the presence of different specific proteases involved in protein digestion was found in A. amygdali using specific substrates and inhibitors. These enzymes had different pH profiles and extracted endogenous inhibitors from the insect’s midgut significantly decreased the majority of the proteases. These results were expected, as A. amyg- dali feeds on several host plants. Sometimes, A. amygdali is used as a rearing host for Trissolcus spp. parasitoids (a biocontrol agent of wheat sunn pest), so these findings could be helpful for efficient mass-rear- ing. A. amygdali occasionally occurs as a secondary pest of agricultural products, so the possible use of protease inhibitors could be a promis- ing method to avoid the use of pesticide sprays. 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