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Digestive proteolytic activity in the pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae)
Journal of Asia-Pacific Entomology 13 (2010) 221–227
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Digestive proteolytic activity in the pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae) Mahdieh Bigham, Vahid Hosseininaveh ⁎ Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31587-77871, Iran
a r t i c l e
i n f o
Article history: Received 23 October 2009 Revised 2 March 2010 Accepted 4 March 2010 Keywords: Brachynema germari Midgut Pistachio Protease Salivary gland
a b s t r a c t Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m1 and m3, respectively, and maximum chymotrypsin and trypsin activities were observed in m3. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010 Published by Elsevier B.V. All rights reserved.
Introduction The pistachio green stink bug, Brachynema germari (Kolenati) (Hemiptera: Pentatomidae), has great importance to pistachio production due to its quantitative and qualitative damages. Serious infestation of nut pistachio by B. germari can make it unpleasant or unmarketable. Piercing of soft-shelled pistachios by the stylets of the stink bug and sucking the nutrients causes epicarp lesion on the hull in the early season (Mehrnejad, 2001), and kernel necrosis in the midseason as well as transmission of the fungal pathogen Nematospora coryli in pistachio nuts (Mehrnejad, 2001; Ershad and Barkhordary, 1976). The injected enzymes digest the nutrients of the nut, especially protein content, and cause direct damage to the kernels. It has been estimated that the total amount of protein in pistachio nuts is about 19.8%, so damaged nuts show considerable reduction in total protein. Foods ingested by insects other than phloem or xylem feeders are mostly macromolecules, such as protein, that have to be broken down by digestive enzymes, such as trypsin (Chapman, 1998). All classes of ⁎ Corresponding author. Department of Plant Protection, College of Agriculture, University of Tehran, P.O. Box: 4111, Karaj 31587-77871, Iran. Tel.: + 98 261 2224022 24 236; fax: + 98 261 2238529. E-mail address: [email protected] (V. Hosseininaveh).
proteolytic enzymes known from vertebrates are also present in insects (Reek et al., 1999). Trypsin-like and chymotrypsin-like serine proteases are commonly found in the midgut of lepidopterans and coleopterans; elastase-like activities, although less common and less abundant, have also been found. Cysteine proteases have been found in slightly acidic midgut juices such as heteropterans and coleopterans (Wieman and Nielsen, 1988). Like the other insects, nutrition and development in green stink bugs is dependent on digestive proteolysis in the gut and salivary gland. Extra oral digestion is facilitated by the secretion of digestive enzymes from the salivary glands (Cohen and Wheeler, 1998). The most frequently reported proteolytic activity in the salivary glands of heteropteran insects is attributed to alkaline proteases (Goodchild, 1952; Hori 1970; Laurema et al., 1985; Cohen, 1990; Hosseininaveh et al., 2009). Proteolytic activity has been detected in salivary glands from many mirids, including Lygus rugulipennis (Laurema et al., 1985) and Creontiades dilutus (Colebatch et al., 2001). In this species, serine proteases are most abundant in the salivary glands (Goodchild, 1952; Hori, 1970; Cohen, 1993). In the predatory bug Zelus renardii, the activity of salivary gland proteases has been further characterized as that of trypsin-like enzymes involved in pre-oral digestion (Cohen, 1993). Acidic proteases, cysteine and aspartic protease, have been detected in the midgut of heteropteran insects (Houseman, 1978; Houseman and Downe, 1982, 1983; Silva and Terra, 1994; Terra, 1988; Cohen, 1993; Terra and
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Ferreira, 1994). Evidence supporting the predominance of acidic proteases includes the acidic nature of the guts of insects within the order Heteroptera (Terra and Ferreira, 1994). Cysteine protease activity, characterized as cathepsin B-like, has been observed in the guts of insects from four families (Houseman, 1978; Houseman et al., 1985), and aspartic proteases has been detected in seven families of the order Heteroptera (Houseman and Downe, 1982, 1983). Currently, one of the most important aspects of pest control is the selective inhibition of the digestive enzymes of many insect pests, because the digestive tract is the main interface region between insects and the environment. In systems of co-evolving plant–insect interactions, plants are able to synthesize a wide range of molecules to defend themselves against insect attack (Bate and Rothstein, 1998). Proteinaceous protease inhibitors (PIs) are active against insect proteolytic enzymes (Ryan, 1990). These inhibitors are insecticidal because they form complexes with digestive enzymes, which are stable and dissociate slowly. Inactivation of digestive enzymes by inhibitors results in blocking of gut proteases, which leads to poor nutrient utilization, retarded development, and death because of starvation (Jongsma and Bolter, 1997; Gatehouse and Gatehouse, 1999). As yet, there is an almost complete lack of knowledge of the digestive proteases in B. germari. This pest's flourishing depends upon their digestive enzymes. In this study, we have reported the morphology of the digestive system, and the detection and classification of digestive proteolytic enzymes present in the midgut and salivary glands of the pistachio green stink bug. Materials and methods Insect B. germari adults (∼ 3000) were collected from a pistachio orchard in Kerman province, Iran. Some insects were immediately dissected, whereas the remainder were maintained on pistachio nuts in the laboratory at 25 ± 1 °C, 55 ± 10% RH and a 16:8 (L:D) photoperiod until needed. Sample preparation Enzyme samples were prepared by the method of Cohen (1993), with slight modifications. The salivary gland complex, including all lobes, accessory glands and tubules was exposed by removal of the prothorax with fine forceps with application of gentle traction to remove the midgut (Zeng et al., 2002). The salivary gland complex and midgut were separated from the insect body, rinsed in ice-cold distilled water and placed in cold distilled water. The salivary gland complex and midgut (including contents) were each homogenized with a hand-held glass grinder on ice. The homogenates were then centrifuged (Hettich universal 32R) at 16,000 (×g) at 4 °C for 10 min. The resulting supernatants were maintained at −20 °C for further use. Alimentary tract and salivary gland complex morphology Adults were dissected in saline solution and divisions between their alimentary tract and salivary gland complex were drawn using a stereomicroscope equipped with a drawing tube. Gut pH determination A series of indicator dyes were used to determine the pH of the gut lumen. The set consisted of neutral red (pH 6.8–8), bromothymol blue (pH 6–7.6), bromocresol purple (pH 5.2–6.8), methyl red (pH 4.4–6.2) and methyl orange (pH 3.1–4.4). The method was based on that of Bignell and Anderson (1980) with slight modifications. Adults were dissected and different divisions of their alimentary tract and salivary
gland complex were removed. Tissues were pooled in 10 µL of distilled water on a microscope slide and broken open with a needle. Aliquots of each pH indicator solutions were then added to the sample and the developed color was compared with the control and recorded. Total protease activity General protease activity of the midgut and salivary gland extracts was determined using bovine hemoglobin as substrate in a broad pH range (pH 4–10). The universal buffer system (40 mM sodium acetate–phosphate–borate) was used to determine the optimum pH of proteolytic activity of midgut and salivary gland complex (Elpidina et al., 2001; Hosseininaveh et al., 2007). The assay was performed according to Cohen (1993) with slight modifications. Hemoglobin solution, 20 mg mL−1, was added to 100 µL of the desired pH universal buffer. Reactions were started with the addition of 40 µL enzyme extract at 30 °C for 120 min. To stop the reaction, 100 µL of 30% trichloroacetic acid (TCA) was added to the reaction mixture. Precipitation was achieved by cooling at 4 °C for 45 min and following centrifugation at 16,000 (×g) for 10 min. TCA was added to the mixture before adding the enzyme extract in blanks. The peptides liberated from hemoglobin were estimated using Folin–Ciocalteo reagent at 630 nm (microplate reader BioTek ELx808) (Folin and Ciocalteu, 1927). Specific protease activity Trypsin and chymotrypsin activities were assayed using a final concentration of 1 mM bz-R-pNA (Nα-benzoyl-L-Arg-p-nitroanilide) and 1 mM s-AAPF-pNA (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) as substrates, respectively. The reaction mixture consisted of 100 µL of enzyme extract, universal buffer of desired pH and 5 µL of the substrates. For cysteine protease assays, two specific substrates, z-RRpNA (benzyloxycarbonyl-Arg-Arg-p-nitroanilide) and z-FR-pNA (benzyloxycarbonyl-Phe-Arg-p-nitroanilide) were used. The absorbance of liberated p-nitroaniline in the reaction mixture was then measured using a microplate reader at 405 nm. All assays were carried out in triplicate, and appropriate blanks were run for all experiments. The effect of pH on proteolytic activity was evaluated by measuring the enzyme activity in different pHs. The substrates bzR-pNA (1 mM), s-AAPF-pNA (1 mM), z-RR-pNA (0.5 mM) and z-FRpNA (0.5 mM) were used for determining proteolytic activity of the midgut extract and bz-R-pNA (1 mM) and z-FR-pNA (0.5 mM) for the salivary gland extract. Effects of inhibitors and activators on proteolytic activity The effect of different inhibitors and activators on proteolytic activities of the midgut and salivary gland complex were determined. The inhibitors used and their concentrations were: Serine protease inhibitors, 1 mM PMSF (phenylmethyl sulfonyl fluoride); trypsin inhibitor, 1 mM TLCK (Nα-p-tosyl-L-lysine chloromethyl ketone); chymotrypsin inhibitor, 1 mM TPCK (N-tosyl-L-phenylalanine chloromethyl ketone); cysteine protease inhibitor, 0.1 µM E-64; and cysteine protease activators, 1 mM L-cysteine and 1 mM DTT (dithiothreitol). To determine the effect of these compounds on enzyme activities, the enzymes were pre-incubated with the inhibitor and activators at room temperature for 15 min, then substrate was added and the assays were carried out as described in the enzyme assay section. Electrophoretic zymogram Electrophoretic detection (FSA Company, Model E1311) of proteolytic enzymes was performed using resolving and stacking polyacrylamide gels of 10% T/0.87%C and 4% T/0.35%C, respectively,
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according to the method described by Garcıa-Carreno et al. (1993) with some modifications. Native-polyacrylamide gel electrophoresis (PAGE) was carried out at 4 °C, and the gels were then immersed in 1% casein in 40 mM sodium acetate–phosphate–borate (pH 6.0) for 120 min at 4 °C to allow the substrate to penetrate into the gel. Then, the gels were transferred to the same solution at 25 °C for 120 min. Finally, the gels were washed in water and immediately fixed and stained with 0.1% coomassie brilliant blue R-250 in a solution of methanol–acetic acid–water (50:10:40). Characterization of protease classes in native-PAGE zymogram by specific inhibitors was then performed. A total of 15 µL of the midgut extract was mixed with 5 µL of E-64 stock solution before electrophoresis. Protein concentration Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Statistical analysis Data were subjected to analysis of variance (ANOVA), and the means were compared by least square difference test (LSD). Statistical analyses were performed using the software Statgraphics Plus 5.1. Results Alimentary tract and salivary gland complex morphology Results showed that the midgut of B. germari, as in other pentatomids, is morphologically divided into four (m1–m4) distinct divisions (Fig. 1). The first section (foremost) of the midgut is dilated, the second is a narrow tube, the third is slightly dilated, and the fourth section (hindmost) is completely different from the other sections because of numerous attached gastric cecae. Salivary glands of the green stink bug showed different sections, including main and accessory glands, the main gland consisting of anterior and posterior lobes (Fig. 2). Determination of the gut pH The midgut of B. germari was found to be slightly acidic. The pH (mean ± SE) was different between the four sections (m1–m4) of the midgut; their pHs were determined to be 6.1 ± 0.2, 6 ± 0.1, 5.9 ± 0.1, 5.9 ± 0.2, respectively (Fig. 1). The pH value of the salivary glands was also determined as slightly acidic, with the value of 5.8 ± 0.2.
Fig. 1. Drawing of multipart midgut of the pistachio green stink bug, B. germari, and their pHs. Midgut is divided into four distinct sections (m1, m2, m3, and m4).
Chymotryptic activity of the midgut extract was exhibited in both acidic and alkaline pH ranges, with two peaks of optimum activities at pHs 4 and 8 (Fig. 3). No considerable tryptic activity was detected in the midgut extract. The midgut extract exhibited maximum hydrolysis of the substrates z-FR-pNA and z-RR-pNA at pHs 5 and 6, respectively (Fig. 4). Maximum hydrolyzing of the substrates bz-RpNA and z-FR-pNA by salivary gland extract was observed at pHs 7–9 and 7–10, respectively (Fig. 5). This result showed that maximum hydrolyzing activity of the salivary glands occurs at alkaline pHs,
Total proteolytic activity The pH dependence of proteolytic activity of the midgut extract from B. germari is shown in Fig. 3. The results showed that the substrate was hydrolyzed over a broad range of acidic (pH = 4–6) and alkaline (pH = 7–10), with a maximum peak of activity at pH 5. No considerable proteolytic activity was detected in salivary gland enzyme extracts when hemoglobin was used as a substrate at different pHs. Specific proteolytic activity Hydrolysis towards the substrates z-FR-pNA and z-RR-pNA (Table 1) suggests the presence of cathepsin L and B in the midgut extract, respectively. Specific substrates for chymotrypsin and trypsin were significantly hydrolyzed by midgut and salivary gland extracts indicating the presence of chymotryptic and tryptic activities in these tissues. No significant hydrolyzing activity was obtained using the substrates z-FR-pNA and z-RR-pNA in the salivary gland extract.
Fig. 2. Drawing of salivary gland complex (anterior and posterior lobes) of B. germari.
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Fig. 3. The effect of pH on the proteolytic activity and chymotryptic activity of midgut extract from B. germari using the substrate hemoglobin. Data are means of triplicate measurements from the same pool of midgut extract with standard errors of the mean.
suggesting that serine proteinases may be the dominant proteases in this organ. Enzyme inhibitors and activators
Fig. 4. The effect of pH on cysteine proteolytic activity (z-FR-pNA for cathepsin L and zRR-pNA for cathepsin B) of the midgut extract from B. germari. Maximum activity takes place at slightly acidic (5 and 6) pHs. Each data point is the mean of triplicate measurements from the same pool of the midgut extract with standard errors of the mean.
specific inhibitor E-64 by the disappearance of the band compared to the control. Compartmentalization of the midgut proteases
Two specific substrates, z-RR-pNA and z-FR-pNA, were used for inhibition assays using specific cysteine protease inhibitor E-64 (1 µm) in the midgut extract. E-64 had high significant inhibition activity towards z-FR-pNA (96 ± 1%) and lower inhibition (38 ± 6%) against the substrate z-RR-pNA. Inhibitory analysis revealed that serine protease activity was partially inhibited by specific serine protease inhibitors. The inhibitors PMSF and TPCK showedsignificant inhibition – 77± 0.5% and 99%, respectively – on chymotryptic activity of the midgut extract. The inhibitors PMSF and TLCK decreased the tryptic activity of the salivary gland up to 82% and 53± 6%, respectively. One of the most important diagnostic features of cysteine proteases is enhanced activity in the presence of thiol compounds (Storey and Wagner, 1986). We examined the effect of DTT and Lcysteine on the hydrolysis activity of the substrates z-FR-pNA and zRR-pNA by midgut extract (Table 2). These substrates were significantly activated by DTT and L-cysteine. The cysteine protease activity of the salivary glands was slightly activated using the activators.
Maximum hydrolysis of z-FR-pNA was obtained in first section of the midgut (m1), but maximum hydrolysis of z-RR-pNA, bz-R-pNA and s-AAPF-pNA was obtained in the third section of the midgut (m3) (Fig. 7). Thus, the maximum cathepsin B activity was detected in m1 and maximum cathepsin L, and chymotryptic and tryptic activities were observed in m3. Discussion Our results revealed that the midgut of B. germari divides into four sections. The first section (m1) is large and saclike, and may serve as a storage region. The second section (m2) serves as a long tube to transfer food material into the third section (m3), where digestion probably occurs. The fourth section (m4) is a slender region
In-gel protease zymogram Further characterization of the protease activity of the midgut extract from B. germari, using casein as substrate in native-PAGE, is shown in Fig. 6. At least one band with protease activity was observed. The cysteine protease activity was confirmed in the presence of the
Table 1 Hydrolytic activity of the midgut and salivary gland extracts against z-RR-pNA (cathepsin B), z-FR-pNA (cathepsin L), s-AAPF-pNA s-AAPF-pNA and bz-R-pNA (trypsin). Substrate
z-RR-pNA z-FR-pNA s-AAPF-pNA bz-R-pNA
Initial velocity (ΔOD min−1) Midgut
Salivary gland
0.003 0.031 0.005 0.001
0.0006 0.001 0.0001 0.001
Fig. 5. The effect of pH on the tryptic and cathepsin L activity (against z-FR-pNA) of salivary gland extract from B. germari. Optimum activity occurs at alkaline pHs. Each datum point is the mean of triplicate measurements from the same pool of salivary gland extract with standard errors of the mean.
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Table 2 Effect of thiol activators (L-cysteine and DTT) on proteolytic activity of the midgut and salivary gland extracts from B. germari. Activator
Enhanced activity % (mean ± SE) Midgut
DTT* L-cystein
Salivary gland
z-RR-pNA (cathepsin B)
z-FR-pNA (cathepsin L)
z-RR-pNA (cathepsin B)
z-FR-pNA (cathepsin L)
118 ± 31 173
446 ± 33 596±21
– 15 ± 0.3
– 18 ± 0.9
*Dithiothreitol.
surrounded with numerous cecae. Multipart-midguts are also observed in various other hemipterans such as Blissus leocopterus (Gillot 2005) and Dysdercus peruvianus (Silva and Terra, 1994). The salivary gland of B. germari consists of a pair of main and accessory glands. The main gland is bilobed and designated as the anterior and posterior lobes, and the accessory glands are tubular. The salivary glands of the pistachio green stink bug are similar to those other hemipterans such as Oncopeltus fasciatus (Chapman 1998) and L. rugulipennis (Laurema et al., 1985). Hemoglobin is routinely used as a conventional substrate for detecting total proteolytic activity (Cohen 1993; Elpidina et al., 2001, Hosseininaveh et al., 2007). Our data show that proteases from midgut extract from B. germari adults are capable of hydrolyzing such a substrate. The pH profile of protease activity using hemoglobin showed the presence of proteases with acidic and alkaline optima in the midgut extract of B. germari. The acidic pH optimum corresponds to cysteine protease activity, whereas the alkaline pH optimum corresponds to serine protease activity. Evidence supporting the predominance of acidic proteases includes the acidic nature of the gut of heteroptera (Terra and Ferreira, 1994). Gut proteases may include not only alkaline proteases, but also other proteases that have optimal activity in neutral or acidic conditions, such as cysteine proteases that have been observed in some coleopteran insects (Michaud et al., 1995; Terra and Cristofoletti, 1996). A bell-shaped curve of total proteolytic activity within a broad range of alkaline pH reveals that B. germari relies on complex proteolytic enzymes for protein digestion. In Hemiptera, protein digestion is usually catalyzed by both cysteine and serine proteases (Colebatch et al., 2001; Zhu et al., 2003). No
Fig. 6. Zymogram analysis of hydrolytic activity of the midgut extract using the substrate casein. The only band disappeared in the presence of the specific cysteine protease inhibitor (E-64).
Fig. 7. Proteolytic activity of m1–m4 extract from B. germari against the substrates z-RRpNA (cathepsin B), z-FR-pNA (cathepsin L), bz-R-pNA (trypsin) and s-AAPF-pNA (chymotrypsin). One unit of enzyme activity (U) is the amount of enzyme capable of liberating 1 µmol of product per minute. The columns with similar letters in different sections are not significantly different (p = 0.05).
significant activity was observed in the salivary gland extract using hemoglobin as substrate. The ability to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors and enhanced activation by thiol compounds revealed the presence of cathepsin L, cathepsin B, chymotrypsin, and slight trypsin activities in the midgut extract. In contrast to the pH for maximum tryptic and chymotryptic activity, the pH of optimal activity of cathepsin B and L is slightly acidic and corresponds to the pH prevailing in the midgut. Optimal trypsin activity was observed at an alkaline pH, whereas chymotrypsin-like proteases are active over a broader range of pH and have two peaks of maximum activities, one at slightly acidic and the other at alkaline pH values. The first peak relates to the pH prevailing in the midgut and may be related to other proteases such as cysteine proteases. Our data show the presence of trypsin and chymotrypsin, albeit in low activity in the salivary glands of B. germari. s-AAPF-pNA hydrolysis was inhibited by PMSF and TPCK in the B. germari, indicating the presence of chymotryptic activity in the midgut of this insect. The presence of the serine proteases in digestive system of hemipterans and other insects is well documented. Serine proteases in Lygus lineolaris are major proteases in the gut tissue and bz-R-pNAase activities in the gut were suppressed by TLCK (Zhu et al., 2003). The midgut extract from Rhodnius prolixus also showed hydrolysis of bz-R-pNA (Houseman and Downe, 1979). Enzymatic action in Tenebrio molitor has demonstrated the presence of high sAAPF-pNAase activity and significant inhibition by PMSF (Elpidina et al., 2005). The synthetic substrates bz-R-pNA and s-AAPF-pNA hydrolyzed by Tribolium castaneum gut proteases and the inhibitors TLCK and TPCK were effective on the gut proteases (Oppert et al., 2003). Different activities against z-RR-pNA and z-FR-pNA suggest the existence of a large amount of cathepsin L and B cysteine proteases. Indeed, our data show that cathepsin L appears to be the major cysteine proteases in the midgut of B. germari, which supports a major role for cathepsin L and a minor role for cathepsin B in the midgut of the pistachio green stink bug. Cathepsin B and L in the posterior midgut of R. prolixus was also determined (Houseman and Downe, 1983). The inhibitor E-64 is commonly used for detection of cysteine proteases in digestive extracts (Colebatch et al., 2001). Using E-64 we demonstrated that cysteine proteases in the pistachio green stink bug could be the major proteases in the midgut extract. Analysis of protease activity in Perillus bioculatus showed that almost 90% of the protease activity was cysteine type using the inhibitor E-64 (Overney
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et al., 1998). Cathepsin L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity in alfalfa weevil midgut, and E-64 causes reduction in total activity (Wilhite et al., 2000). Proteolytic activity reported in the gut of L. lineolaris had an optimum pH of 4.25 and was postulated to be a cysteine protease, based on inhibition by E-64 (Wright et al., 2006). Digestive proteolytic activities of the pistachio green stink bug were significantly enhanced by thiol compounds such as DTT and L-cysteine confirming the presence of cysteine proteases. The hydrolysis of the gut extract in Lissorhoptrus brevirostris and D. peruvianus was also activated by DTT and L-cysteine (Silva and Terra, 1994; Hernandez et al., 2003). Proteolysis of the gut extract was activated by 1.5 mM DTT in Callosobruchus maculates and Zabrotes subfasciatus (Silva et al., 2001). In general, chymotrypsin and cathepsin B and L, like cysteine proteases, are mainly located in the midgut of the pistachio green stink bug. Cysteine protease activity seems to be higher than serine proteases activities in midgut extract of B. germari. Similarly, among phytophagous Hemiptera, the cotton stainer, D. peruvianus, also uses cysteine protease for protein digestion (Silva and Terra, 1994). Organization of digestion in m1-m4 parts of the midgut is different. The major part of cathepsin B activity was located in the first section. In some insects, such as Acyrthosiphon pisum, most cysteine protease activity occurs in m1 (Cristofoletti et al., 2003). In L. brevirostris, the level of cathepsin B activity in the anterior and middle section of the gut are two to three times and more than 10 times higher than in the posterior section, respectively (Hernandez et al., 2003). The acidic pH medium of the midgut implies that acidic proteases predominate, which is consistant with the activity of cysteine proteases. Considerable tryptic activity and slight chymotryptic activity in present the salivary glands of the pistachio green stink bug. The tryptic activity of these extracts was definitely detected at alkaline pHs, which is not consistent with the pH of the salivary glands. Significant inhibition of protease activity of the salivary gland extract by PMSF and TLCK indicates that major protease activity in the salivary glands resulted from serine proteases, especially trypsin-like proteases. Serine protease activities have been also detected in the salivary glands of a very closely-related species, Eurygaster integriceps (Hosseininaveh et al., 2009). Trypsin-like serine protease activities were found in L. lineolaris and L. hesperus based on hydrolysis of bz-RpNA and inhibition by PMSF (Zeng et al., 2002; Zhu et al., 2003). Evidence of a trypsin-like enzyme has also been found in the salivary gland of Deraeocoris nigritulus (Boyd 2003). Salivary enzymes are an important component of digestion in predatory Heteroptera (Cohen 1990), and the extent of pre-oral digestion in phytophagous Heteroptera is unclear (Colebatch, 2001). Weak cysteine protease activity in the salivary glands of the pistachio green stink bug indicates a minor role of these enzymes in digestion. Proteolytic activity was slightly enhanced by thiol compounds in the salivary gland extract, but not to a significant degree in comparison with those of the midgut extract. No cysteine protease activity was detected in L. lineolaris (Wright et al., 2006). No cysteine protease gene was identified from the salivary glands of the green mirid, suggesting that this class of protease is not expressed in this tissue (Colebatch et al., 2002). The acid protease in the salivary glands of Lygus could be lysosomal cathepsin D acid proteases in insect tissues during metamorphosis (Kawamura et al., 1984). Therefore, we concluded that alkaline proteases predominate in the salivary glands for protein digestion in B. germari. In this study, we observed differences in the relative activities of proteases from the midgut extract and those from the salivary gland of B. germari. The existence of both cysteine and serine proteases in the midgut of the pistachio green stink bug suggests a complex mix of proteases may be contributing to digestion in this insect, perhaps also due to the multipart differentiation of the midgut (Goodchild 1963). Noticeable proteolytic activity occurred in the third part of the midgut, and this part may be the major part of digestion in the pistachio green
stink bug. This kind of enzyme compartmentalization is also observed in other insects, such as L. brevirostris (Hernandez et al., 2003). References Bate, N.J., Rothstein, S.J., 1998. C6-Volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes. Plant J. 16, 561–569. Bignell, D.E., Anderson, J.M., 1980. Determinaion of pH and oxygen status in the guts of lower and higher termites. J. Insect Physiol. 26, 183–188. Boyd, D.W., 2003. Digestive enzyme and stylet morphology of Deraeocoris nigritulus (Uher) (Hemiptera: Miridae) refelect adaptation for predatory habits. Ann. Entomol. Soc. Am. 96, 667–671. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Chapman, R.F., 1998. The Insect: Structure and Function, 4th ed. Cambridge University Press. 770 pp. Cohen, A.C., 1990. Feeding adaptations of some predaceous Hemiptera. Ann. 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Midgut adaptation and digestive enzyme distribution in a phloem feeding insect the pea aphid Acyrthosiphon pisum. J. Insect physiol. 49, 11–24. Elpidina, E.N., Vinokurov, K.S., Gromenko, V.A., Rudenskaya, Y.A., Dunaevsky, Y.E., Zhuzhikov, D.P., 2001. Compartmentalization of proteases and amylases in Nauphoeta cinerea midgut. Arch. Insect Biochem. Physiol. 48, 206–216. Elpidina, E.N., Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A., Zhuzhikov, D.P., Oppert, B., 2005. A chymotrypsin-like protease from the midgut of Tenebrio molitor larvae. Biochimie 87, 771–779. Ershad, D., Barkhordary, M., 1976. Investigations on stigmatomycosis (massu disease) of pistachio. J. Plant Pathol. 12, 17–23. Folin, O., Ciocalteu, V., 1927. On tyrosine and tryptophane determination in proteins. J. Biol. Chem. 73, 627–650. Garcıa-Carreno, F.L., Dimes, N., Haard, N., 1993. Substrategel electrophoresis for composition and molecular weight of proteases or proteinaceous protease inhibitors. Anal. 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Entomol. Zool. 5, 51–61. Hosseininaveh, V., Bandani, A.R., Azmayeshfard, P., Hosseinkhani, S., Kazzazi, M., 2007. Digestive proteolytic and amylolytic activities in Trogoderma granarium Everts (Dermestidae: Coleoptera). J. Stored Prod. Res. 43, 515–522. Hosseininaveh, V., Bandani, A.R., Hosseininaveh, F., 2009. Digestive proteolytic activity in the Sunn pest, Eurygaster integriceps. J. Insect Sc. 9, 70. Houseman, J., 1978. A thiol activated digestive protease from adults of Rhodinus prolixus stal (Hemiptera: Reduviidae). Can. J. Zool. 56, 1140–1143. Houseman, J.G., Downe, A.E.R., 1982. Characterization of an acidic protease from the posterior midgut of Rhodnius prolixus stal (Hemiptera: Reduviidae). Insect Biochem. 12, 51–655. Houseman, J.G., Downe, A.E.R., 1983. Cathepsin D-like activity in the posterior midgut of hemipteran insects. Comp. Biochem. Physiol. 75B, 509–512. Houseman, J.G., Morrison, P.E., Downe, A.E.R., 1985. Cathepsin B and aminopeptidase in the posterior midgut of phymata wolffii stal (Hemiptera: Phymatidae). Can. J. Zool. 63, 1288–1291. Jongsma, M.A., Bolter, C., 1997. The adaptation of insect to plant protease inhibitors. J. Insect Physiol. 43, 885–896. Kawamura, M., Wadano, A., Miura, K., 1984. Purification and some properties of Cathepsin-like thiol protease from pupae of the blow fly Aldrichina grahami. Comp. Biochem. Physiol. 78B, 279–286. Laurema, S., Varis, A., Miettinen, H., 1985. Studies on enzymes in the salivary glands of Lygus ruglipennis (Hemiptera, Miridae). Insect Biochem. 15, 211–224. Mehrnejad, M.R., 2001. The current status of pistachio pests in Iran. Cah. Opt. Med. 56, 315–322.
M. Bigham, V. Hosseininaveh / Journal of Asia-Pacific Entomology 13 (2010) 221–227 Michaud, D., Bernier-Vadnais, N., Overney, S., Yelle, S., 1995. Constitutive expression of digestive cysteine protease forms during development of the Colorado potato beetle, Leptinotarsa decemlineata say (Coleptera: Chrysomelidae). Insect Biochem. Mol. Biol. 25, 1041–1048. Oppert, B., Morgan, T.D., Hartzert, K., Lenarich, B., Galesa, K., Brizin, J., Turk, V., Yoza, K., Ohtsubo, K., Kramer, K.J., 2003. Proteases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. 134, 481–490. Overney, S., Yelle, S., Cloutier, C., 1998. Occurrence of digestive cysteine proteases in Perillus bioculatus, a natural predator of the Colorado potato beetls. Comp. Biochem. Physiol. 20B, 191–195. Reek, G., Oppert, B., Denton, M., Kanost, M., Baker, J.E., Kramer, K.J., 1999. Insect proteases. In: Turk, V. (Ed.), Proteases: New Persective. Birkhauser Verlag, Basel, Boston, pp. 125–148. Silva, C.P., Terra, W.R., 1994. Digestive and absorptive sites along midgut of the cotton seed sucker bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae). Insect Biochem. Mol. Biol. 25, 493–505. Silva, C.P., Terra, W.R., Lima, R.M., 2001. Difference in midgut serine proteases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus. Arch. Insect Biochem. Physiol. 47, 18–28. Storey, R.D., Wagner, F.W., 1986. Plant proteases: a need for uniformity. Phytochem. 25, 2701–2709.
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Terra, W.R., 1988. Physiology and biochemistry of insect digestion: an evolutionary perspective. Braz. J. Med. Biol. Res. 21, 675–734. Terra, W.R., Cristofoletti, P.T., 1996. Midgut protease in three divergent species of Coleoptera. Comp. Biochem. Physiol. 113B, 725–730. Terra, W.R., Ferreira, C., 1994. Insect digestive enzymes: properties, compartmentalization and function. Comp. Biochem. Physiol. 109B, 1–62. Wieman, K.F., Nielsen, S.S., 1988. Isolation and partial characterization of a major gut protease from larval Acanthoscelides obtectus Say (Coleopteran: Bruchidae). Comp. Biochem. Physiol. 89B, 419–426. Wilhite, S.E., Elden, T.C., Brzin, J., Smigocki, A.C., 2000. Inhibition of cysteine and aspartyl proteases in the alfalfa weevil midgut with biochemical and plant-derived protease inhibitors. Insect Biochem. Mol. Biol. 30, 1181–1188. Wright, M.K., Brandt, S.L., Coudron, T.A., Wagner, R.M., Habibi, J., Backus, E.A., Huesing, J. E., 2006. Characterization of digestive proteolytic activity in Lygus hesperus knight (Hemiptera: Miridae). J. Insect Physiol. 52, 717–728. Zeng, F., Zhu, Y., Cohen, A.C., 2002. Partial characterization of trypsin-like-protease and molecular cloning of a trypsin-like precursor cDNA in salivary glands of Lygus lineolaris. Comp. Biochem. Physiol. 131, 453–463. Zhu, Y.C., Zeng, F., Oppert, B., 2003. Molecular cloning of trypsin-like cDNAs and comparison of protease activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae). Insect Biochem. Mol. Biol. 33, 889–899.
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Digestive proteolytic activity in the pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae) Mahdieh Bigham, Vahid Hosseininaveh ⁎ Department of Plant Protection, College of Agriculture, University of Tehran, Karaj 31587-77871, Iran
a r t i c l e
i n f o
Article history: Received 23 October 2009 Revised 2 March 2010 Accepted 4 March 2010 Keywords: Brachynema germari Midgut Pistachio Protease Salivary gland
a b s t r a c t Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m1 and m3, respectively, and maximum chymotrypsin and trypsin activities were observed in m3. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010 Published by Elsevier B.V. All rights reserved.
Introduction The pistachio green stink bug, Brachynema germari (Kolenati) (Hemiptera: Pentatomidae), has great importance to pistachio production due to its quantitative and qualitative damages. Serious infestation of nut pistachio by B. germari can make it unpleasant or unmarketable. Piercing of soft-shelled pistachios by the stylets of the stink bug and sucking the nutrients causes epicarp lesion on the hull in the early season (Mehrnejad, 2001), and kernel necrosis in the midseason as well as transmission of the fungal pathogen Nematospora coryli in pistachio nuts (Mehrnejad, 2001; Ershad and Barkhordary, 1976). The injected enzymes digest the nutrients of the nut, especially protein content, and cause direct damage to the kernels. It has been estimated that the total amount of protein in pistachio nuts is about 19.8%, so damaged nuts show considerable reduction in total protein. Foods ingested by insects other than phloem or xylem feeders are mostly macromolecules, such as protein, that have to be broken down by digestive enzymes, such as trypsin (Chapman, 1998). All classes of ⁎ Corresponding author. Department of Plant Protection, College of Agriculture, University of Tehran, P.O. Box: 4111, Karaj 31587-77871, Iran. Tel.: + 98 261 2224022 24 236; fax: + 98 261 2238529. E-mail address: [email protected] (V. Hosseininaveh).
proteolytic enzymes known from vertebrates are also present in insects (Reek et al., 1999). Trypsin-like and chymotrypsin-like serine proteases are commonly found in the midgut of lepidopterans and coleopterans; elastase-like activities, although less common and less abundant, have also been found. Cysteine proteases have been found in slightly acidic midgut juices such as heteropterans and coleopterans (Wieman and Nielsen, 1988). Like the other insects, nutrition and development in green stink bugs is dependent on digestive proteolysis in the gut and salivary gland. Extra oral digestion is facilitated by the secretion of digestive enzymes from the salivary glands (Cohen and Wheeler, 1998). The most frequently reported proteolytic activity in the salivary glands of heteropteran insects is attributed to alkaline proteases (Goodchild, 1952; Hori 1970; Laurema et al., 1985; Cohen, 1990; Hosseininaveh et al., 2009). Proteolytic activity has been detected in salivary glands from many mirids, including Lygus rugulipennis (Laurema et al., 1985) and Creontiades dilutus (Colebatch et al., 2001). In this species, serine proteases are most abundant in the salivary glands (Goodchild, 1952; Hori, 1970; Cohen, 1993). In the predatory bug Zelus renardii, the activity of salivary gland proteases has been further characterized as that of trypsin-like enzymes involved in pre-oral digestion (Cohen, 1993). Acidic proteases, cysteine and aspartic protease, have been detected in the midgut of heteropteran insects (Houseman, 1978; Houseman and Downe, 1982, 1983; Silva and Terra, 1994; Terra, 1988; Cohen, 1993; Terra and
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2010.03.004
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Ferreira, 1994). Evidence supporting the predominance of acidic proteases includes the acidic nature of the guts of insects within the order Heteroptera (Terra and Ferreira, 1994). Cysteine protease activity, characterized as cathepsin B-like, has been observed in the guts of insects from four families (Houseman, 1978; Houseman et al., 1985), and aspartic proteases has been detected in seven families of the order Heteroptera (Houseman and Downe, 1982, 1983). Currently, one of the most important aspects of pest control is the selective inhibition of the digestive enzymes of many insect pests, because the digestive tract is the main interface region between insects and the environment. In systems of co-evolving plant–insect interactions, plants are able to synthesize a wide range of molecules to defend themselves against insect attack (Bate and Rothstein, 1998). Proteinaceous protease inhibitors (PIs) are active against insect proteolytic enzymes (Ryan, 1990). These inhibitors are insecticidal because they form complexes with digestive enzymes, which are stable and dissociate slowly. Inactivation of digestive enzymes by inhibitors results in blocking of gut proteases, which leads to poor nutrient utilization, retarded development, and death because of starvation (Jongsma and Bolter, 1997; Gatehouse and Gatehouse, 1999). As yet, there is an almost complete lack of knowledge of the digestive proteases in B. germari. This pest's flourishing depends upon their digestive enzymes. In this study, we have reported the morphology of the digestive system, and the detection and classification of digestive proteolytic enzymes present in the midgut and salivary glands of the pistachio green stink bug. Materials and methods Insect B. germari adults (∼ 3000) were collected from a pistachio orchard in Kerman province, Iran. Some insects were immediately dissected, whereas the remainder were maintained on pistachio nuts in the laboratory at 25 ± 1 °C, 55 ± 10% RH and a 16:8 (L:D) photoperiod until needed. Sample preparation Enzyme samples were prepared by the method of Cohen (1993), with slight modifications. The salivary gland complex, including all lobes, accessory glands and tubules was exposed by removal of the prothorax with fine forceps with application of gentle traction to remove the midgut (Zeng et al., 2002). The salivary gland complex and midgut were separated from the insect body, rinsed in ice-cold distilled water and placed in cold distilled water. The salivary gland complex and midgut (including contents) were each homogenized with a hand-held glass grinder on ice. The homogenates were then centrifuged (Hettich universal 32R) at 16,000 (×g) at 4 °C for 10 min. The resulting supernatants were maintained at −20 °C for further use. Alimentary tract and salivary gland complex morphology Adults were dissected in saline solution and divisions between their alimentary tract and salivary gland complex were drawn using a stereomicroscope equipped with a drawing tube. Gut pH determination A series of indicator dyes were used to determine the pH of the gut lumen. The set consisted of neutral red (pH 6.8–8), bromothymol blue (pH 6–7.6), bromocresol purple (pH 5.2–6.8), methyl red (pH 4.4–6.2) and methyl orange (pH 3.1–4.4). The method was based on that of Bignell and Anderson (1980) with slight modifications. Adults were dissected and different divisions of their alimentary tract and salivary
gland complex were removed. Tissues were pooled in 10 µL of distilled water on a microscope slide and broken open with a needle. Aliquots of each pH indicator solutions were then added to the sample and the developed color was compared with the control and recorded. Total protease activity General protease activity of the midgut and salivary gland extracts was determined using bovine hemoglobin as substrate in a broad pH range (pH 4–10). The universal buffer system (40 mM sodium acetate–phosphate–borate) was used to determine the optimum pH of proteolytic activity of midgut and salivary gland complex (Elpidina et al., 2001; Hosseininaveh et al., 2007). The assay was performed according to Cohen (1993) with slight modifications. Hemoglobin solution, 20 mg mL−1, was added to 100 µL of the desired pH universal buffer. Reactions were started with the addition of 40 µL enzyme extract at 30 °C for 120 min. To stop the reaction, 100 µL of 30% trichloroacetic acid (TCA) was added to the reaction mixture. Precipitation was achieved by cooling at 4 °C for 45 min and following centrifugation at 16,000 (×g) for 10 min. TCA was added to the mixture before adding the enzyme extract in blanks. The peptides liberated from hemoglobin were estimated using Folin–Ciocalteo reagent at 630 nm (microplate reader BioTek ELx808) (Folin and Ciocalteu, 1927). Specific protease activity Trypsin and chymotrypsin activities were assayed using a final concentration of 1 mM bz-R-pNA (Nα-benzoyl-L-Arg-p-nitroanilide) and 1 mM s-AAPF-pNA (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) as substrates, respectively. The reaction mixture consisted of 100 µL of enzyme extract, universal buffer of desired pH and 5 µL of the substrates. For cysteine protease assays, two specific substrates, z-RRpNA (benzyloxycarbonyl-Arg-Arg-p-nitroanilide) and z-FR-pNA (benzyloxycarbonyl-Phe-Arg-p-nitroanilide) were used. The absorbance of liberated p-nitroaniline in the reaction mixture was then measured using a microplate reader at 405 nm. All assays were carried out in triplicate, and appropriate blanks were run for all experiments. The effect of pH on proteolytic activity was evaluated by measuring the enzyme activity in different pHs. The substrates bzR-pNA (1 mM), s-AAPF-pNA (1 mM), z-RR-pNA (0.5 mM) and z-FRpNA (0.5 mM) were used for determining proteolytic activity of the midgut extract and bz-R-pNA (1 mM) and z-FR-pNA (0.5 mM) for the salivary gland extract. Effects of inhibitors and activators on proteolytic activity The effect of different inhibitors and activators on proteolytic activities of the midgut and salivary gland complex were determined. The inhibitors used and their concentrations were: Serine protease inhibitors, 1 mM PMSF (phenylmethyl sulfonyl fluoride); trypsin inhibitor, 1 mM TLCK (Nα-p-tosyl-L-lysine chloromethyl ketone); chymotrypsin inhibitor, 1 mM TPCK (N-tosyl-L-phenylalanine chloromethyl ketone); cysteine protease inhibitor, 0.1 µM E-64; and cysteine protease activators, 1 mM L-cysteine and 1 mM DTT (dithiothreitol). To determine the effect of these compounds on enzyme activities, the enzymes were pre-incubated with the inhibitor and activators at room temperature for 15 min, then substrate was added and the assays were carried out as described in the enzyme assay section. Electrophoretic zymogram Electrophoretic detection (FSA Company, Model E1311) of proteolytic enzymes was performed using resolving and stacking polyacrylamide gels of 10% T/0.87%C and 4% T/0.35%C, respectively,
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according to the method described by Garcıa-Carreno et al. (1993) with some modifications. Native-polyacrylamide gel electrophoresis (PAGE) was carried out at 4 °C, and the gels were then immersed in 1% casein in 40 mM sodium acetate–phosphate–borate (pH 6.0) for 120 min at 4 °C to allow the substrate to penetrate into the gel. Then, the gels were transferred to the same solution at 25 °C for 120 min. Finally, the gels were washed in water and immediately fixed and stained with 0.1% coomassie brilliant blue R-250 in a solution of methanol–acetic acid–water (50:10:40). Characterization of protease classes in native-PAGE zymogram by specific inhibitors was then performed. A total of 15 µL of the midgut extract was mixed with 5 µL of E-64 stock solution before electrophoresis. Protein concentration Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Statistical analysis Data were subjected to analysis of variance (ANOVA), and the means were compared by least square difference test (LSD). Statistical analyses were performed using the software Statgraphics Plus 5.1. Results Alimentary tract and salivary gland complex morphology Results showed that the midgut of B. germari, as in other pentatomids, is morphologically divided into four (m1–m4) distinct divisions (Fig. 1). The first section (foremost) of the midgut is dilated, the second is a narrow tube, the third is slightly dilated, and the fourth section (hindmost) is completely different from the other sections because of numerous attached gastric cecae. Salivary glands of the green stink bug showed different sections, including main and accessory glands, the main gland consisting of anterior and posterior lobes (Fig. 2). Determination of the gut pH The midgut of B. germari was found to be slightly acidic. The pH (mean ± SE) was different between the four sections (m1–m4) of the midgut; their pHs were determined to be 6.1 ± 0.2, 6 ± 0.1, 5.9 ± 0.1, 5.9 ± 0.2, respectively (Fig. 1). The pH value of the salivary glands was also determined as slightly acidic, with the value of 5.8 ± 0.2.
Fig. 1. Drawing of multipart midgut of the pistachio green stink bug, B. germari, and their pHs. Midgut is divided into four distinct sections (m1, m2, m3, and m4).
Chymotryptic activity of the midgut extract was exhibited in both acidic and alkaline pH ranges, with two peaks of optimum activities at pHs 4 and 8 (Fig. 3). No considerable tryptic activity was detected in the midgut extract. The midgut extract exhibited maximum hydrolysis of the substrates z-FR-pNA and z-RR-pNA at pHs 5 and 6, respectively (Fig. 4). Maximum hydrolyzing of the substrates bz-RpNA and z-FR-pNA by salivary gland extract was observed at pHs 7–9 and 7–10, respectively (Fig. 5). This result showed that maximum hydrolyzing activity of the salivary glands occurs at alkaline pHs,
Total proteolytic activity The pH dependence of proteolytic activity of the midgut extract from B. germari is shown in Fig. 3. The results showed that the substrate was hydrolyzed over a broad range of acidic (pH = 4–6) and alkaline (pH = 7–10), with a maximum peak of activity at pH 5. No considerable proteolytic activity was detected in salivary gland enzyme extracts when hemoglobin was used as a substrate at different pHs. Specific proteolytic activity Hydrolysis towards the substrates z-FR-pNA and z-RR-pNA (Table 1) suggests the presence of cathepsin L and B in the midgut extract, respectively. Specific substrates for chymotrypsin and trypsin were significantly hydrolyzed by midgut and salivary gland extracts indicating the presence of chymotryptic and tryptic activities in these tissues. No significant hydrolyzing activity was obtained using the substrates z-FR-pNA and z-RR-pNA in the salivary gland extract.
Fig. 2. Drawing of salivary gland complex (anterior and posterior lobes) of B. germari.
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Fig. 3. The effect of pH on the proteolytic activity and chymotryptic activity of midgut extract from B. germari using the substrate hemoglobin. Data are means of triplicate measurements from the same pool of midgut extract with standard errors of the mean.
suggesting that serine proteinases may be the dominant proteases in this organ. Enzyme inhibitors and activators
Fig. 4. The effect of pH on cysteine proteolytic activity (z-FR-pNA for cathepsin L and zRR-pNA for cathepsin B) of the midgut extract from B. germari. Maximum activity takes place at slightly acidic (5 and 6) pHs. Each data point is the mean of triplicate measurements from the same pool of the midgut extract with standard errors of the mean.
specific inhibitor E-64 by the disappearance of the band compared to the control. Compartmentalization of the midgut proteases
Two specific substrates, z-RR-pNA and z-FR-pNA, were used for inhibition assays using specific cysteine protease inhibitor E-64 (1 µm) in the midgut extract. E-64 had high significant inhibition activity towards z-FR-pNA (96 ± 1%) and lower inhibition (38 ± 6%) against the substrate z-RR-pNA. Inhibitory analysis revealed that serine protease activity was partially inhibited by specific serine protease inhibitors. The inhibitors PMSF and TPCK showedsignificant inhibition – 77± 0.5% and 99%, respectively – on chymotryptic activity of the midgut extract. The inhibitors PMSF and TLCK decreased the tryptic activity of the salivary gland up to 82% and 53± 6%, respectively. One of the most important diagnostic features of cysteine proteases is enhanced activity in the presence of thiol compounds (Storey and Wagner, 1986). We examined the effect of DTT and Lcysteine on the hydrolysis activity of the substrates z-FR-pNA and zRR-pNA by midgut extract (Table 2). These substrates were significantly activated by DTT and L-cysteine. The cysteine protease activity of the salivary glands was slightly activated using the activators.
Maximum hydrolysis of z-FR-pNA was obtained in first section of the midgut (m1), but maximum hydrolysis of z-RR-pNA, bz-R-pNA and s-AAPF-pNA was obtained in the third section of the midgut (m3) (Fig. 7). Thus, the maximum cathepsin B activity was detected in m1 and maximum cathepsin L, and chymotryptic and tryptic activities were observed in m3. Discussion Our results revealed that the midgut of B. germari divides into four sections. The first section (m1) is large and saclike, and may serve as a storage region. The second section (m2) serves as a long tube to transfer food material into the third section (m3), where digestion probably occurs. The fourth section (m4) is a slender region
In-gel protease zymogram Further characterization of the protease activity of the midgut extract from B. germari, using casein as substrate in native-PAGE, is shown in Fig. 6. At least one band with protease activity was observed. The cysteine protease activity was confirmed in the presence of the
Table 1 Hydrolytic activity of the midgut and salivary gland extracts against z-RR-pNA (cathepsin B), z-FR-pNA (cathepsin L), s-AAPF-pNA s-AAPF-pNA and bz-R-pNA (trypsin). Substrate
z-RR-pNA z-FR-pNA s-AAPF-pNA bz-R-pNA
Initial velocity (ΔOD min−1) Midgut
Salivary gland
0.003 0.031 0.005 0.001
0.0006 0.001 0.0001 0.001
Fig. 5. The effect of pH on the tryptic and cathepsin L activity (against z-FR-pNA) of salivary gland extract from B. germari. Optimum activity occurs at alkaline pHs. Each datum point is the mean of triplicate measurements from the same pool of salivary gland extract with standard errors of the mean.
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Table 2 Effect of thiol activators (L-cysteine and DTT) on proteolytic activity of the midgut and salivary gland extracts from B. germari. Activator
Enhanced activity % (mean ± SE) Midgut
DTT* L-cystein
Salivary gland
z-RR-pNA (cathepsin B)
z-FR-pNA (cathepsin L)
z-RR-pNA (cathepsin B)
z-FR-pNA (cathepsin L)
118 ± 31 173
446 ± 33 596±21
– 15 ± 0.3
– 18 ± 0.9
*Dithiothreitol.
surrounded with numerous cecae. Multipart-midguts are also observed in various other hemipterans such as Blissus leocopterus (Gillot 2005) and Dysdercus peruvianus (Silva and Terra, 1994). The salivary gland of B. germari consists of a pair of main and accessory glands. The main gland is bilobed and designated as the anterior and posterior lobes, and the accessory glands are tubular. The salivary glands of the pistachio green stink bug are similar to those other hemipterans such as Oncopeltus fasciatus (Chapman 1998) and L. rugulipennis (Laurema et al., 1985). Hemoglobin is routinely used as a conventional substrate for detecting total proteolytic activity (Cohen 1993; Elpidina et al., 2001, Hosseininaveh et al., 2007). Our data show that proteases from midgut extract from B. germari adults are capable of hydrolyzing such a substrate. The pH profile of protease activity using hemoglobin showed the presence of proteases with acidic and alkaline optima in the midgut extract of B. germari. The acidic pH optimum corresponds to cysteine protease activity, whereas the alkaline pH optimum corresponds to serine protease activity. Evidence supporting the predominance of acidic proteases includes the acidic nature of the gut of heteroptera (Terra and Ferreira, 1994). Gut proteases may include not only alkaline proteases, but also other proteases that have optimal activity in neutral or acidic conditions, such as cysteine proteases that have been observed in some coleopteran insects (Michaud et al., 1995; Terra and Cristofoletti, 1996). A bell-shaped curve of total proteolytic activity within a broad range of alkaline pH reveals that B. germari relies on complex proteolytic enzymes for protein digestion. In Hemiptera, protein digestion is usually catalyzed by both cysteine and serine proteases (Colebatch et al., 2001; Zhu et al., 2003). No
Fig. 6. Zymogram analysis of hydrolytic activity of the midgut extract using the substrate casein. The only band disappeared in the presence of the specific cysteine protease inhibitor (E-64).
Fig. 7. Proteolytic activity of m1–m4 extract from B. germari against the substrates z-RRpNA (cathepsin B), z-FR-pNA (cathepsin L), bz-R-pNA (trypsin) and s-AAPF-pNA (chymotrypsin). One unit of enzyme activity (U) is the amount of enzyme capable of liberating 1 µmol of product per minute. The columns with similar letters in different sections are not significantly different (p = 0.05).
significant activity was observed in the salivary gland extract using hemoglobin as substrate. The ability to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors and enhanced activation by thiol compounds revealed the presence of cathepsin L, cathepsin B, chymotrypsin, and slight trypsin activities in the midgut extract. In contrast to the pH for maximum tryptic and chymotryptic activity, the pH of optimal activity of cathepsin B and L is slightly acidic and corresponds to the pH prevailing in the midgut. Optimal trypsin activity was observed at an alkaline pH, whereas chymotrypsin-like proteases are active over a broader range of pH and have two peaks of maximum activities, one at slightly acidic and the other at alkaline pH values. The first peak relates to the pH prevailing in the midgut and may be related to other proteases such as cysteine proteases. Our data show the presence of trypsin and chymotrypsin, albeit in low activity in the salivary glands of B. germari. s-AAPF-pNA hydrolysis was inhibited by PMSF and TPCK in the B. germari, indicating the presence of chymotryptic activity in the midgut of this insect. The presence of the serine proteases in digestive system of hemipterans and other insects is well documented. Serine proteases in Lygus lineolaris are major proteases in the gut tissue and bz-R-pNAase activities in the gut were suppressed by TLCK (Zhu et al., 2003). The midgut extract from Rhodnius prolixus also showed hydrolysis of bz-R-pNA (Houseman and Downe, 1979). Enzymatic action in Tenebrio molitor has demonstrated the presence of high sAAPF-pNAase activity and significant inhibition by PMSF (Elpidina et al., 2005). The synthetic substrates bz-R-pNA and s-AAPF-pNA hydrolyzed by Tribolium castaneum gut proteases and the inhibitors TLCK and TPCK were effective on the gut proteases (Oppert et al., 2003). Different activities against z-RR-pNA and z-FR-pNA suggest the existence of a large amount of cathepsin L and B cysteine proteases. Indeed, our data show that cathepsin L appears to be the major cysteine proteases in the midgut of B. germari, which supports a major role for cathepsin L and a minor role for cathepsin B in the midgut of the pistachio green stink bug. Cathepsin B and L in the posterior midgut of R. prolixus was also determined (Houseman and Downe, 1983). The inhibitor E-64 is commonly used for detection of cysteine proteases in digestive extracts (Colebatch et al., 2001). Using E-64 we demonstrated that cysteine proteases in the pistachio green stink bug could be the major proteases in the midgut extract. Analysis of protease activity in Perillus bioculatus showed that almost 90% of the protease activity was cysteine type using the inhibitor E-64 (Overney
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et al., 1998). Cathepsin L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity in alfalfa weevil midgut, and E-64 causes reduction in total activity (Wilhite et al., 2000). Proteolytic activity reported in the gut of L. lineolaris had an optimum pH of 4.25 and was postulated to be a cysteine protease, based on inhibition by E-64 (Wright et al., 2006). Digestive proteolytic activities of the pistachio green stink bug were significantly enhanced by thiol compounds such as DTT and L-cysteine confirming the presence of cysteine proteases. The hydrolysis of the gut extract in Lissorhoptrus brevirostris and D. peruvianus was also activated by DTT and L-cysteine (Silva and Terra, 1994; Hernandez et al., 2003). Proteolysis of the gut extract was activated by 1.5 mM DTT in Callosobruchus maculates and Zabrotes subfasciatus (Silva et al., 2001). In general, chymotrypsin and cathepsin B and L, like cysteine proteases, are mainly located in the midgut of the pistachio green stink bug. Cysteine protease activity seems to be higher than serine proteases activities in midgut extract of B. germari. Similarly, among phytophagous Hemiptera, the cotton stainer, D. peruvianus, also uses cysteine protease for protein digestion (Silva and Terra, 1994). Organization of digestion in m1-m4 parts of the midgut is different. The major part of cathepsin B activity was located in the first section. In some insects, such as Acyrthosiphon pisum, most cysteine protease activity occurs in m1 (Cristofoletti et al., 2003). In L. brevirostris, the level of cathepsin B activity in the anterior and middle section of the gut are two to three times and more than 10 times higher than in the posterior section, respectively (Hernandez et al., 2003). The acidic pH medium of the midgut implies that acidic proteases predominate, which is consistant with the activity of cysteine proteases. Considerable tryptic activity and slight chymotryptic activity in present the salivary glands of the pistachio green stink bug. The tryptic activity of these extracts was definitely detected at alkaline pHs, which is not consistent with the pH of the salivary glands. Significant inhibition of protease activity of the salivary gland extract by PMSF and TLCK indicates that major protease activity in the salivary glands resulted from serine proteases, especially trypsin-like proteases. Serine protease activities have been also detected in the salivary glands of a very closely-related species, Eurygaster integriceps (Hosseininaveh et al., 2009). Trypsin-like serine protease activities were found in L. lineolaris and L. hesperus based on hydrolysis of bz-RpNA and inhibition by PMSF (Zeng et al., 2002; Zhu et al., 2003). Evidence of a trypsin-like enzyme has also been found in the salivary gland of Deraeocoris nigritulus (Boyd 2003). Salivary enzymes are an important component of digestion in predatory Heteroptera (Cohen 1990), and the extent of pre-oral digestion in phytophagous Heteroptera is unclear (Colebatch, 2001). Weak cysteine protease activity in the salivary glands of the pistachio green stink bug indicates a minor role of these enzymes in digestion. Proteolytic activity was slightly enhanced by thiol compounds in the salivary gland extract, but not to a significant degree in comparison with those of the midgut extract. No cysteine protease activity was detected in L. lineolaris (Wright et al., 2006). No cysteine protease gene was identified from the salivary glands of the green mirid, suggesting that this class of protease is not expressed in this tissue (Colebatch et al., 2002). The acid protease in the salivary glands of Lygus could be lysosomal cathepsin D acid proteases in insect tissues during metamorphosis (Kawamura et al., 1984). Therefore, we concluded that alkaline proteases predominate in the salivary glands for protein digestion in B. germari. In this study, we observed differences in the relative activities of proteases from the midgut extract and those from the salivary gland of B. germari. The existence of both cysteine and serine proteases in the midgut of the pistachio green stink bug suggests a complex mix of proteases may be contributing to digestion in this insect, perhaps also due to the multipart differentiation of the midgut (Goodchild 1963). Noticeable proteolytic activity occurred in the third part of the midgut, and this part may be the major part of digestion in the pistachio green
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