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Biochemical characterization of digestive proteases and carbohydrases of the carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)
Abstract
Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.
... We showed that trypsin serine protease is main specific protease in larvae P. xylostella which is in accordance with other studies (Naseri et al., 2010;Tabatabaei et al., 2011). Although chymotrypsin and elastase were also found in P. xylostella their activity was not great enough to be detected by zymogram. ...
... The larvae feeding on SLM046 and Green-Cornet had more tryptic activity than those feeding on RGS003 and Globe-Master. Existence of various isoforms for trypsin enzyme has been reported for other lepidopteran insects (Tabatabaei et al., 2011). ...
... By studying rate of glucosides hydrolase and biochemical properties of these enzymes in P. xylostella and their response to P. xylostella feeding on different plant hosts, we found that the high activity of α-and β-galactosidase occurred on susceptible cultivars which is in line with other reports (Terra and Ferriera, 1994;Azevedo et al., 2003). The optimal acidity for α-glucosidase and β-glucosidase activities were at pH 6.0 and 7.0, respectively, showing the glucosidase group is more efficient at slightly acidic to neutral pH such as that seen in some lepidopteran insects (Frandsen and Svensson, 1998;Tabatabaei et al., 2011). ...
The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most destructive insect pests, feeding exclusively on wild and cultivated cruciferous species. The attacked plants produce considerable amount of glucosinolates in response to insects' feeding. Herein, we studied digestive activities of P. xylostella on four different genotypes of family Brassicaceae including two canola cultivars (SLM046 and RGS003) and two cabbage cultivars (Green-Cornet and Glob-Master). The highest proteolytic and amylolytic activities of P. xylostella were observed on Green-Cornet and the lowest occurred on RGS003 and Glob-Master, respectively. The highest activity of α-glucosidase and β-glucosidases were observed on Green-Cornet and SLM046 and the lowest was observed on Glob-Master and RGS003. The zymogram analysis revealed different isozymes of protease, trypsin-like and α-amylase in the midgut extract of P. xylostella. Activity of the above mentioned isozymes was inhibited in larvae feeding on RGS003 and Glob Master as resistant host cultivars. Also, larvae feeding on the resistant genotypes showed more glucosidase activities, indicating possibility of high glycosinolate existence in the resistant genotypes. By these results we can state that host plant property can affect insect digestive physiology through inhibiting digestive enzyme activities. These findings provide insights into the direct effects of host plants on insect physiology which are conducive to change in insect fitness.
... (MATTIACCI et al. 1995). Studying the biochemical characteristics of the CPB's digestive enzymes is a necessary prerequisite for developing cultivars resistant to CPB (RAZAVI TABATABAEI et al. 2011). α-Glucosidase (EC 3.2.1.20) ...
... The purification of α-and β-glucosidase and studies of their enzymatic activity under different conditions have been done on many different insect species (SANTOS & TERRA 1985, PRATVIEL et al. 1987, MARANA et al. 1995, SILVA et al. 1999, MARANA et al. 2000, PONTOH & LOW 2002, TOKUDA et al. 2002, AZEVEDO et al. 2003, CARNEIRO et al. 2004, BYEON et al. 2005, SOUZA-NETO et al. 2007, RAMZI & HOSSEININAVEH 2010, RAZAVI TABATABAEI et al. 2011. Considering the importance of carbohydrases and the study of carbohydrate digestion as a target for L. decemlineata control, the aim of this study is to provide information about some biochemical properties of α-and β-glucosidase extracted from the midgut of L. decemlineata in order to acquire a better understanding of the digestive physiology of the CPB. ...
... The electropherogram analysis of the L. decemlineata midgut showed single and three distinct bands for α-and β-glucosidase respectively. In previous studies with different insect species, α-glucosidase produced at least two (RAZAVI TABATABAEI et al. 2011) or three distinct bands (SILVA et al. 1999). Furthermore, in the midgut of many insect species there are three, four or even five digestive β-glucosidases (AZEVEDO et al. 2003). ...
Host plant resistance is an environmentally safe method used for reducing a pest population. Basically, when developing resistant cultivars one needs to study the biochemical characteristics of the digestive enzymes in the insect’s midgut. In this study, the activities of α- and β-glucosidase were determined from Leptinotarsa decemlineata midgut using p-nitrophenyl-α-Dglucopyranoside and p-nitrophenyl-β-D-glucopyranoside as substrates respectively. The results showed that the specific activity of α- and β-glucosidase from 4th instar larvae midguts of L. decemlineata were 5.14 and 5.48 Umg
... Proteases are very important enzymes in insects, as they hydrolyze the peptide bonds in dietary proteins to liberate the amino acids needed for growth, survival, and reproduction, and because they detoxify protein toxins ingested as a consequence of plant and pathogen feeding (Terra et al. 1996). The lepidopteran larvae need a proteolytic enzyme complex including trypsins, chymotrypsins, elastases, cathepsin-B like proteases, aminopeptidases, and carboxypeptidases for protein digestion, and many serine proteases are dominant in the larval gut (Patankar et al. 2001;Srinivasan et al. 2006;Chougule et al. 2008;Tabatabaei et al. 2011). Since there is significant variation among the biochemical properties of insect digestive proteases, their characterization is necessary for designing a safe control strategy that utilizes plant-proteinaceous inhibitors (Wilhite et al. 2000). ...
... Alkaline pH values for activity are due to intrinsic alkaline pH of the insect digestive system, and have been reported for many lepidopteran insects ( Purcell et al. 1992;Ferry et al. 2005;Budatha et al. 2008;Chougule et al. 2008). Tabatabaei et al. (2011) showed that proteolytic activity of the carob moth, Ectomyelois ceratoniae, with hemoglobin as protein substrate occurred over a broad alkaline pH range (pH 8.0-11.0), with maximum activity at pH 10. ...
... Midgut proteinases of E. ceratoniae hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8.0-11.0 (Tabatabaei et al. 2011). The pH activity profile of the trypsin-like in G. pyloalis is different from that of other lepidopteran larvae, which show a pH optimum below 11.0 (Ahmed et al. 1980;Johnston et al. 1991Johnston et al. , 1995Purcell et al. 1992;Gatehouse et al. 1999). ...
Abstract Proteolytic activities in digestive system extracts from the larval midgut of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), were analyzed using different specific peptide substrates and proteinase inhibitors. High proteolytic activities were found at pH 10.0 and a temperature of 50° C using azocasein as substrate. The trypsin was active in the pH range of 9.5- 12.0, with its maximum activity at pH 11.5. Ethylene diamine tetraacetic acid had the most inhibitory effect, and 44% inhibition was detected in the presence of this inhibitor. Phenyl methane sulfonyl floride and N-tosyl-L-phe chloromethyl ketone also showed considerable inhibition of larval azocaseinolytic activity, with 40.2 and 35.1% inhibition respectively. These data suggest that the midgut of larvae contains mainly metalloproteases and serine proteases, mainly chymotrypsin. The effect of several metal ions on the activity of proteases showed that NaCl, CaCl2, CoCl2 (5 and 10 mM), and MnCl2 (5mM) reduced the protease activity. The kinetic parameters of trypsin-like proteases using N-benzoyl-L-arg-p-nitroanilide as substrate indicated that the Km and Vmax values of trypsin in the alimentary canal were 50.5 ± 2.0 µM and 116.06 ± 1.96 nmol min(-1) mg(-1) protein, respectively. Inhibition assays showed only small amounts of cysteine proteases were present in the G. pyloalis digestive system. The midgut digestive protease system of G. pyloalis is as diverse as that of any of the other polyphagous lepidopteran insect species, and the midgut of larvae contains mainly metalloproteases. Moreover, serine proteases and chymotrypsin also play main roles in protein digestion. Characterization of the proteolytic properties of the digestive enzymes of G. pyloalis offers an opportunity for developing appropriate and effective pest management strategies via metalloproteases and chymotrypsin inhibitors.
... The method of Tabatabaei et al. (2011) was used for α-amylase assays. Briefly, α-amylase (EC 3.2.1.1) ...
... High levels of organic acids observed in PA could simultaneously lead to a reduction in pH in the gut lumen. Generally, the lumen pH of most lepidopteran midguts is alkaline, around 9-11 (Terra and Ferreira 1994; Tabatabaei et al., 2011); thus, consuming substances that reduce pH is likely to have impacts on the activity levels of digestive enzymes (Gringorten et al., 1993;Terra et al., 1996;Pytelkova et al., 2009). Reduced activity levels of digestive enzymes can lead to a reduced absorption of nutrients that are necessary to for the growth, development, and reproductive success of herbivorous insects (Stygar et al., 2010). ...
Natural products derived from plant materials, such as wood vinegar (pyroligneous acid; PA), have shown toxicity to insects in previous studies; however, the impact of this substance on physiology and life history of stored product insects is unknown. Thus, the current study aimed to determine the effects of PA on two prominent stored product moths, Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). LC50/24h values of PA were determined for both species and first instar larvae were fed on an artificial diet containing LC50 concentrations of PA for their entire life cycle. Final instar larvae were selected for biochemical analysis while life history parameters were assayed in female adults that emerged from treated larvae. In both species, significantly lower activities of digestive enzymes (α-amylase, protease, and lipase) and detoxifying enzymes (carboxylesterase, monooxygenase cytochrome P450, and glutathione S-transferase) were observed in treated larvae relative to untreated larvae. In contrast, antioxidant enzyme activities (superoxide dismutase, phenoloxidase, and ascorbate peroxidase) were significantly elevated in treated larvae compare to controls in both species. Moreover, larvae of both species tended to have lower levels of energy reserves, such as proteins, lipids, and carbohydrates, and adults exhibited reduced longevity, fecundity, and oviposition periods in comparison to untreated larvae. Finally, GC-MS analysis of the PA used for this study demonstrated that phenolic compounds and organic acids were the largest constituents. These results indicate that consumption of PA leads to the induction of to the antioxidant system, the inhibition of detoxification and digestion enzyme activities as well as reduced levels of energy reserves. Cumulatively, these effects resulted in reduced longevity and fecundity compared to control insects. This study expands our knowledge of the impacts of PA on physiology and life history for two prominent pests of stored products.
... Also, in this study it was found that gut of E. ceratoniae larvae contains three different α-amylase isoforms, similar to what has been found in others insects [25, 35-37, 32, 38] , that presence of three isoforms is a strategy to escape from inhibitory toxicity [39] , and also it indicates the importance of carbohydrates in its diet. Our results show that the α-amylase in E. ceratoniae larvae have an alkaline optimal pH, which is consistent with the optimal pH that has been reported by Tabatabaei et al. (2011) [40] . In the present study, it was found that seed proteinaceous extracts of six plant species including amaranth, barley, pea, bean, mung bean and wild oat affected α-amylase activity of the insect gut. ...
... Also, in this study it was found that gut of E. ceratoniae larvae contains three different α-amylase isoforms, similar to what has been found in others insects [25, 35-37, 32, 38] , that presence of three isoforms is a strategy to escape from inhibitory toxicity [39] , and also it indicates the importance of carbohydrates in its diet. Our results show that the α-amylase in E. ceratoniae larvae have an alkaline optimal pH, which is consistent with the optimal pH that has been reported by Tabatabaei et al. (2011) [40] . In the present study, it was found that seed proteinaceous extracts of six plant species including amaranth, barley, pea, bean, mung bean and wild oat affected α-amylase activity of the insect gut. ...
The carob moth is a cosmopolitan pest of many agricultural commodities both in the field and in the
storage. The aim of the current study was to investigate the effects of seed proteinaceous extracts against
E. ceratoniae α-amylase. The results showed that bean, wild oat, pea, mung bean, barley and amaranth
inhibited the enzyme activity with the percentage of 17, 26, 32, 37, 51 and 71 respectively. Also, when
different concentrations of amaranth and barley extracts were assayed against α-amylase, a concentration
dependent trend was observed. Also, amaranth and barley seed extracts had an optimum pH and
temperature inhibition of 10 and 35 °C which are the optimum pH and temperature for the activity of this
enzyme in the in vitro condition. Thus, it is concluded that amaranth seed proteins are potentially good
for detailed investigation in order to get a clear picture of its active compound(s) and its structurefunction
relationship.
... In this context, insect gut can act as a promising option for isolating proteases that hold industrially important characteristics. The insect gut contains several proteases such as trypsins, chymotrypsins, cathepsin-b like proteases, aminopeptidases, elastases, carboxypeptidases and serine proteases, which are responsible for the hydrolysis of leaf organic matter, and synthesis of small peptides and amino acids for its growth and development ( Applebaum, 1985;Patankar et al., 2001;Srinivasan et al., 2006;Chougule et al., 2008;Tabatabaei et al., 2011). The insect gut proteases are considered as a target for insect pest management and industrial application because of their unusual potential to function in the alkaline microenvironment of the gut (pH 10.0-12.0) ...
... For the past twenty years, several researchers studied larval gut enzymes and its inhibitory mechanism for insect pest control ( Srinivasan et al., 2006;Chougule et al., 2008;Tabatabaei et al., 2011). Very few studies have focused on insect gut protease and its significance in various industrial applications ( Mika et al., 2013). ...
In the present study, one-dimensional gel electrophoresis (1-DE) and two-dimensional gel electrophoresis (2-DE) coupled zymography were performed for pupal gut proteins which revealed a single prominent proteolytic band/spot at molecular weight of 37 kDa and an isoelectric point (pI) at 5 to 6 approximately. The proteolytic spot was identified as 37 k-protease of B. mori using mass spectrometry. This 37k-Pupal gut serine protease (PGSP) was purified by anionic exchange and gel filtration chromatography. The biochemical characteristics of PGSP were evaluated using quantitative protease assay. PGSP has substrate specificity with gelatin and cannot be inhibited by various protease inhibitors. However, it was sensitive to reducing agents, dithiothreitol and β-mercaptoethanol. The proteolytic activity was not affected by metal ions (10 mM) except Cu²⁺ and Hg²⁺. The optimum pH and temperature of PGSP was determined as pH 9.0 and 60 °C. The purified protease revealed significant stability and compatibility towards surfactants, oxidizing agent and commercial detergents at 25% concentration. The PGSP (90 µg) with 1% detergent (Rin) showed complete destaining of blood stained cloth. It has significant clot lysis (> 40%) activity within 3 h. In addition, the PGSP shows stable activity in presence of 25% concentration of organic solvents. The overall results suggest that PGSP of B. mori could be utilized as an additive agent in detergent industry.
... The membrane was then removed, and placed for 5 min in 0.1% sodium nitrite, 0.5% ammonium sulfamate, and 0.05% N-(1-naphthyl) ethylenediamine solutions, so that the bands would appear. The effect of plant inhibitors on the tryptic activity was evaluated based on the method of Razavi Tabatabaei et al. (2011). The enzymes and inhibitors were briefly mixed together and incubated for 30 min. ...
... Previous research on zymogram of trypsin in the digestive system of lepidopteran larva showed that there were diverse isoforms. For example, a zymogram study of Ectomyelois ceratoniae (Zeller) revealed that this insect has 4 isoforms of trypsin (Razavi Tabatabaei et al. 2011). Whereas, the zymogram pattern of H. armigera showed 20 isoforms of trypsin activity (Srinivasan et al. 2006). ...
Proteases are one of the most important digestive enzymes in the midgut of Hyphantria cunea Drury. Proteases are responsible for protein digestion. In the present study, we evaluated the efficiency of some plant inhibitors on proteases in the gut of the H. cunea. Last instar larvae were collected from mulberry trees. The digestive system of the larvae was used as an enzyme source. The total proteolytic and trypsin activity were assessed by the hemoglobin and BApNA, respectively, as the substrate. The evaluation of the total proteolytic and trypsin activities in various pHs showed the highest relative activity at a pH of 11. Also, the inhibitory effect of inhibitors extracted from Alhagi maurorum Medik., Lathyrus sativus L., Vicia faba L., Prosopis farcta (Banks & Sol.) Eig., and Panicum miliaceum L. on the digestive protease of the fall webworm was measured. Protease inhibitors extracted from A. maurorum, P. farcta and P. miliaceum showed negligible inhibition but L. sativus was able to inhibit 34.72% and 100% of the total activity of proteolytic and trypsin, respectively. Also, the total proteolytic and trypsin activities were inhibited by the inhibitor from V. faba, at 22.27% and 100%, respectively. The zymogram pattern of trypsin with nitro-cellulose membranes showed 2 isoforms in the gut of H. cunea. The inhibitor from L. sativus completely inhibited both isoforms. Gel electrophoresis of proteolitytic activity revealed at least 6 isoforms the inhibitor extracted from L. sativus; completely inhibiting some of them. The inhibitor from L. sativus was purified by ammonium sulfate precipitation and gel-filtration. The molecular mass of the inhibitor was determined as 45 kDa. The highest inhibition of trypsin activity by the inhibitor from L. sativus occurred at a pH of 10. The stability of the inhibitor from L. sativus was evaluated at different pHs and temperatures. The results showed that the inhibitor from L. sativus was stable at a pH of 11.0, and showed 45% inhibition on trypsin activity at a pH of 11. Also, this inhibitor revealed stability up to 50°C.
... Detoxification and antioxidant enzyme assays were performed using extracts from the whole body of larvae or newly emerged worker bees (Zhang et al., 2012) as follows: Glutathione S-transferase (GST) by the method of Roy et al. (2021), acetylcholine esterase (AChE) by the method in Shahriari et al. (2018), alpha and beta carboxylesterases (1-CaE and 2-CaE, respectively) by the method of Zibaee et al. (2009), superoxide dismutase (SOD) by the method in Krishnan et al. (2002), and ascorbate peroxidase (APX) by the method described by Shamakhi et al. (2018). Digestive enzymes were assayed in gut samples isolated from larvae or newly emerged worker bees (Masoumzadeh et al., 2014) as follows: a-amylase based on the method of Tabatabaei et al. (2011) and protease by the method of Ramzi et al. (2013). Energy reserves were evaluated using wholebody homogenates of larvae and adult worker bees. ...
... The complex proteolytic enzymes used by lepidopteran larvae for protein digestion include trypsins, chymotrypsins, elastases, cathepsin-B-like proteases, aminopeptidases, and carboxypeptidases (Chougule et al., 2008;Patankar et al., 2001). The gut of the larvae is dominated by several serine proteases (Tabatabaei et al., 2011). Serine proteases are the predominant approach to food digestion in lepidopteran insects and account for roughly 95% of all digestive action (Srinivasan et al., 2006;Chougule et al., 2008). ...
The cotton bollworm larva, Pectinophora gossypiella is a threat to cotton crops globally. The pest attack result in qualitative and quantitative loss. Gut proteases of pest are studied to target its digestive system for inhibition. The biochemical and electrophoretic properties of proteases from the digestive system of Pectinophora gossypiella were determined. By using a well-diffusion assay, it was confirmed that P. gossypiella gut extract contained proteases. The proteolytic activity obtained in the solution assay was 0.116 Umg. The impact of varying pH and temperatures on proteolytic activity was assessed. At pH 10 and a temperature of 50°C, it showed that proteolytic activity was at its peak. To see the isoform of proteases in gel, native PAGE gelatine zymography was used. There are a total of 8 protease isoforms in all instars. The total protein profile of P. gossypiella was also determined. There is currently no information available about the digestive proteases of P. gossypiella. Detailed insights into the gut proteases of P. gossypiella can be used to create and enhance successful pest management techniques based on protease inhibitors.
... The major proteases in the larval gut of lepidopteran insects belong to the serine family (e.g. trypsin and chymotrypsin), and a few others belong to the aspartic, metallo and cysteine protease family [96][97][98]. The in vitro inhibition assays along with IC 50 values obtained with purified BBIs/KIs revealed that VrBBI and CpBBI are effective in inhibiting the gut proteases of A. janata whereas VrKI and CpKI are effective in inhibiting the gut proteases of H. armigera ( Fig. 9A and B, Table 3). ...
Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (˂24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.
... Most of these site-specific release systems were inspired from drug delivery and food science research, whereas systems that promote agricultural release applications are still at an early stage. A few papers have reported that enzymes present in the salivary glands and midgut of larvae and insects are good candidates for triggering the release owing to the presence of carbohydrates, glycans and proteases [54][55][56][57][58]. For example, Oliveira et al. [58] showed that carrier systems based on zein nanoparticles (loaded with botanical Fig. 2 The nano-pesticide lunch-box principle. ...
Nature contains many examples of “fake promises” to attract “prey”, e.g., predatory spiders that emit the same sex-attractant-signals as moths to catch them at close range and male spiders that make empty silk-wrapped gifts in order to mate with a female. Nano-pesticides should ideally mimic nature by luring a target and killing it without harming other organisms/species. Here, we present such an approach, called the lunch-box or deadly-goodies approach. The lunch-box consists of three main elements (1) the lure (semio-chemicals anchored on the box), (2) the box (palatable nano-carrier), and (3) the kill (advanced targeted pesticide). To implement this approach, one needs to draw on the vast amount of chemical ecological knowledge available, combine this with recent nanomaterial techniques, and use novel advanced pesticides. Precision nano-pesticides can increase crop protection and food production whilst lowering environmental impacts.
Graphical Abstract
... Various proteases have been identified in the midgut of Lepidoptera larvae, including trypsin, chymotrypsin, elastases, cathepsin B-like proteases, aminopeptidases and carboxypeptidases (Chougule et al., 2008;Terra and Ferreira, 2012). Serine protease activity (particularly trypsin and chymotrypsin) accounts for 95% of the total protease activity in the midgut of these caterpillars (Chougule et al., 2008;Srinivasan et al., 2006;Tabatabaei et al., 2011). Proteases are present in multiple isoforms whose expression can be altered to accomplish optimal protein digestion (Patankar et al., 2001;Srinivasan et al., 2006). ...
Cadmium (Cd) presence in terrestrial ecosystems is a serious threat that requires continuous development of biomonitoring tools. Ideally, a suitable biomarker of exposure should respond to the toxicant consistently in different populations regardless of previous exposure to pollution. Here we considered the activities and isoform patterns of certain proteases and acid phosphatases (ACP) in the midgut of Lymantria dispar larvae as well as the integrated biomarker response (IBR) for application in Cd biomonitoring. We compared the responses of caterpillars originating from unpolluted and polluted localities after they had been chronically subjected to dietary Cd (50 and 100 μg Cd/g dry food). The population inhabiting the unpolluted forest was far more sensitive to Cd exposure as the activities of total proteases, trypsin (TRY) and leucine aminopeptidase (LAP) were mostly reduced while the activities of total and non-lysosomal ACP were increased. Non-lysosomal ACP activity was elevated in larvae from the contaminated site in response to the higher Cd concentration. Exposure to the metal resulted in numerous alterations in the pattern of enzyme isoforms, but the responses of the two populations were similar except that larvae from the polluted locality were more tolerant to the lower Cd concentration. Non-lysosomal ACP activity and the appearance of ACP isoforms 4 and 5 together with the IBR index are the most promising indicators of Cd presence, potentially applicable even in populations with a history of exposure to pollution. TRY and total ACP activities could be used to monitor populations at uncontaminated localities.
... In the midgut of Lepidoptera protease activity mainly originates from serine proteases, such as trypsin which hydrolyzes internal peptide bonds of the polypeptide chain on the carboxyl side of basic L-amino acids. They contribute up to 95% of the total digestive activity (Terra and Ferreira, 1994;Srinivasan et al., 2006;Tabatabaei et al., 2011;Yao et al., 2012). Aminopeptidases are metalloenzymes that remove amino acid residues from the N-terminus of peptide chains (Valaitis, 1995). ...
Fluoranthene is one of the most abundant polycyclic aromatic hydrocarbon pollutants in the environment and it may accumulate in plant leaves which are the main food source for phytophagous insect species. The aim of this study was to establish the effects of dietary fluoranthene on specific activities of digestive enzymes and expression of their isoforms in the midgut, and the relative growth rates of Lymantria dispar and Euproctis chrysorrhoea larvae. Exposure to fluoranthene led to significantly decreased trypsin activity in the midgut of larvae of both species. Leucine aminopeptidase activity decreased significantly in the midgut of L. dispar larvae exposed to the lower concentration of fluoranthene, but that enzyme activity showed the opposite trend in E. chrysorrhoea larvae. There was no pollutant induced changes in lipase activity in L. dispar, while elevated enzyme activity was recorded in the midgut of E. chrysorrhoea larvae exposed to the lower concentration of fluoranthene. Different patterns of expression of enzyme isoforms were noticed. Relative growth rates of both species significantly decreased in fluoranthene treated larvae. These responses indicate to the significance of relationships between physiological changes and fitness-related traits in L. dispar and E. chrysorrhoea larvae affected by pollutant, and contribute to understanding the mechanisms of their adjustment to stressful conditions.
... Special attention has been focused on enzymes present in the salivary glands and mid-gut of larvae and insects, in the soil, and produced by phytopathogenic fungi. The salivary glands and mid-gut of insects mainly contain carbohydrases and proteases [15,56,57]. In soil, the enzymes most frequently found are urease, alkaline phosphatase, dehydrogenase, and catalase [2]. ...
Abstract Pesticides and fertilizers are widely used to enhance agriculture yields, although the fraction of the pesticides applied in the field that reaches the targets is less than 0.1%. Such indiscriminate use of chemical pesticides is disadvantageous due to the cost implications and increasing human health and environmental concerns. In recent years, the utilization of nanotechnology to create novel formulations has shown great potential for diminishing the indiscriminate use of pesticides and providing environmentally safer alternatives. Smart nano-based pesticides are designed to efficiently delivery sufficient amounts of active ingredients in response to biotic and/or abiotic stressors that act as triggers, employing targeted and controlled release mechanisms. This review discusses the current status of stimuli-responsive release systems with potential to be used in agriculture, highlighting the challenges and drawbacks that need to be overcome in order to accelerate the global commercialization of smart nanopesticides.
... Serine proteases isolated from Lepidoptera midguts show clear alkaline pH optima, probably as a consequence of adaptations to the alkaline midgut environment (Christeller et al. 1992). Most of the remaining proteolytic activity in Lepidoptera midguts is executed by cathepsin-B like cysteine proteases, and different exopeptidases (Christeller et al. 1992;Terra et al. 1994;Bown et al. 1997;Patankar et al. 2001;Breugelmans et al. 2009;Tabatabaei et al. 2011). The midguts of Orthoptera are characterized by a neutral pH, and in parallel, the majority of proteolytic activity in the midguts of this order results from serine proteases (Chapman 2013). ...
Insects are the most abundant and diverse class of animals on the planet. One explanation for their success is their extraordinary ability to successfully consume a wide range of foods. Like all heterotrophic organisms, insects need to acquire vital nutrients from their diet. The central organ for food digestion and absorption of nutrients is the gastrointestinal tract. This organ’s principal functions are mediating the efficient digestion of the diet and protecting the organism against harmful chemicals, microorganisms, and mechanical damage from the food. These functions are achieved through regional differentiation of the alimentary canal as well as highly flexible adaptations to the consumed diets, both at anatomical and molecular levels. Numerous studies describing the general gut morphology and associated digestive mechanisms of various insects exist. Nevertheless, the molecular patterns underlying digestion and nutrient uptake in insects are still poorly characterized. This review aims to provide an overview of the general strategies of extracellular macronutrient digestion and consequent nutrient absorption found among different orders of insects.
... Till date, insect gut proteases are of main interest among the industrial biotechnologists because of their unusual alkaline micro-environment with midgut pH ranging from 10 to 12 (Christeller et al., 1992). Therefore, insect gut can be used as a promising source for isolating industrially important enzymes (Ahmad et al., 1980;Patankar et al., 2001;Saleemuddin, 2000, 2002;Srinivasan et al., 2006;Chougule et al., 2008;Parde et al., 2012;Tabatabaei et al., 2011;Sanatan et al., 2013;Visweshwar et al., 2015). Akbar and Sharma (2017) recommended the use of insect as bio-resource for isolation of insect gut protease due to the short life cycle, voracious feeding of different host plants and production of diverse multiple enzymes (trypsin, chymotrypsin, elastase, cathepsin, aminopeptidase, carboxypeptidase and many serine proteases) from the gut with rapid catalytic and chemo-stable activity. ...
Recently, insect gut protease has gained great interest in thefield of food and industrial biotechnology due totheir invisible characteristics and also their ability to act as an alternative source for microbial protease. Theinsect gut proteases are produced either by themselves or by gut symbiotic microbes, that utilize it for theirmetabolism. In this review, the importance of insect gut proteases was highlighted in terms of general physio-chemical properties (pH, temperature and metal) and their compatibility with detergents and resistance tosolvents with broad applications in various industries such as laundry detergents, bio-medical, food industry andbio-ethanol production. The production of insect gut protease can be increased through emerging biotechno-logical techniques to meet out the demand for protease in future.
... Functional group present in the active site of protease leads to be categorized four classes of protease i.e. serine protease, aspartic protease, cysteine protease, and metallo protease (Rao et al., 1998). Trypsin, chymotrypsin, and elastase-like enzymes belonging to serine protease category play a vital role in the digestion of protein in the gut of insects (Srinivasan et al., 2006;Chougule, 2008;Tabatabaei et al., 2011).The antagonistic molecules generally termed as protease inhibitor (PI) molecules with wider occurrence (microbes, plants, and animals) are responsible for the inhibition of these proteases and could be a key element in insect resistance transgenic technology (Ryan, 1900). ...
The adaptability of insect pest to prolong chemical insecticides is gradually incising problem facing worldwide. Environmental impacts and human health concern point towards the need of an ecofriendly alternative to insect pest control. Insect gut proteases are supposed to be involved in protein digestion. As well these are believed to be involved in adaptive contrivances exerted by host plants. Protease helps to digest proteinaceous diet acquired from host plant in the insect gut. As a counter response host plants secrets protease inhibitors molecules to resist the proteolytic action of insect gut proteases. The Helicoverpa armigera is major insect pest of legumes mainly pigeon pea, chick pea, Soybean etc. It affects and causes serious damage and significant yield lost. The predominant serine protease activity in the gut is observed. These proteases are responsible for degradation of host protease inhibitor molecules and protein digestion in the gut. Due to the presence of complex protease system this insect pest is found to be adapting on various host plants. The digging into interactions between these two molecules i.e. insect gut protease and protease inhibitor of host plants is necessary to design future strategies to manage this insect pest in agricultural crops. In this regard, we made a hypothesis that the peptides released during degradation of protease inhibitors of host plants are responsible for further inhibition of gut proteases. To ascertain our hypothesis at the primary level here we in silico analyzed interaction patterns of serine protease of H. armigera with randomly fragmented PI peptides of BBI of host plants. The serine protease structure modeled for sequence UniProt ID O18447. Secondary structure prediction by the SOPMA server revealed that random coil (34.25%) dominated among secondary structure elements and alpha helices (33.86%), extended strand (19.69%) and beta turn (12.20%). The Ramchandran plot generated by PROCHECK validate the structure modeled through UCSF Chimera modeler to be of high quality with 81.8%. The accuracy of 3D folding of protein by Verify3D plot showed that more than 83% of the residues had a positive 3D-1D averaged score. COACH gives higher C-score: 0.98 denote a more reliable prediction of the catalytic site. The virtual screening of fragment library explores that Bowman-Birk inhibitor has two potent active domains which have the ability to block serine protease. The BBI fragments C41-F50 and D56-S65 give higher affinity score -8.3 for both, As compared with whole ligand binding with active site residues. Further, Molecular dynamics gives a stable interaction results. Autodock 4.0 and Vina used to inspect hypothesis as well as molecular dynamics did for validation by Gromacs. Natural compound libraries from ZINC Page VIII screen with the novel structure of protease. Where ZINC08214665 and ZINC38140521 found a potent inhibitor for serine protease with the binding affinity (-10.9) and (-9.5) respectively. ZINC38140521 is Maslinic acid which is derived as a byproduct from the processing of dry olive-pomace oil. Many in vitro studies show that Maslinic acid inhibits serine protease and use as a key enzyme for the controlling HIV. Here we are reporting new aspect for Masculine acid as a natural inhibitor for insect gut protease. The countered hypothesis is proved and showing a new way for the entomologist to in silico approach is beneficial to study insect gut enzymes with host plant inhibitors. Here we firstly report an interaction between randomly fragmented stretches of amino acids with H.armigera gut serine protease. Further study of the interaction between sitespecific fragmented active stretches of amino acids with protease catalytic site and wet-lab confirmation is the future aspect.
... The 70 gene members identified in the serine-protease family pointed out an essential role in the spider mite physiology (Grbic et al., 2011). In many phytophagous insects, particularly in lepidopteran, the participation of serine proteases in the gut digestion has been demonstrated (Lara et al., 2000;Patankar et al., 2001;Fan and Wu, 2005;Srinivasan et al., 2006;Chougule et al., 2008;Tabatabaei et al., 2011). However, the lack of detection of trypsin-and chymotrypsin activities in mite extracts (Carrillo et al., 2011) together with the fact that serine-protease genes did not show a clear developmental pattern of expression correlated with feeding stages (Santamaria et al., , 2015b, and the absence of this activity in mite feces suggested the association of this protease type with physiological processes other than the hydrolysis of dietary proteins (Santamaria et al., 2015b). ...
Tetranychus urticae (two-spotted spider mite) is a striking example of polyphagy among herbivores with an extreme record of pesticide resistance and one of the most significant pests in agriculture. The T. urticae genome contains a large number of cysteine- and serine-proteases indicating their importance in the spider mite physiology. This work is focused on the potential role of the Kunitz trypsin inhibitor (KTI) family on plant defense responses against spider mites. The molecular characterization of two of these genes, AtKTI4 and AtKTI5, combined with feeding bioassays using T-DNA insertion lines for both genes was carried out. Spider mite performance assays showed that independent KTI silencing Arabidopsis lines conferred higher susceptibility to T. urticae than WT plants. Additionally, transient overexpression of these inhibitors in Nicotiana benthamiana demonstrated their ability to inhibit not only serine- but also cysteine-proteases, indicating the bifunctional inhibitory role against both types of enzymes. These inhibitory properties could be involved in the modulation of the proteases that participate in the hydrolysis of dietary proteins in the spider mite gut, as well as in other proteolytic processes.
... Insects feeding on stored products, rich in carbohydrates, generally have higher amylase activities than those feeding on wool or plants, but the latter usually have higher proteolytic activity (Chapman, 1998). Similar situation has been reported for other lepidopteran and coleopteran pests of stored products such as Plodia interpunctella Hübner (Chapman, 1998), Phthorimaea operculella Zeller (Mansouri et al., 2013), E. kuehniella (Abdi et al. 2014), Trogoderma granarium Everts (Hosseininaveh et al., 2007), and Ectomyelois ceratoinae Zeller (Razavi Tabatabaei et al., 2011). Polysaccharides, especially starch, is one of the major nutrients in plant tissues that must initially be digested by α-amylase to smaller parts for absorption via epithelial cells (Zibaee, 2012). ...
The Mediterranean flour moth, Ephestia kuehniella Zeller is one of the conventional hosts for rearing of natural enemies to be used in biological control programs. In this study, the effects of three cereal flours (wheat, corn and barley) on some biological parameters, nutritional responses, as well as proteolytic and amylolytic digestive activities of the fifth instar larvae of E. kuehniella were studied to determine suitability of the cereals for optimum growth and reproduction of E. kuehniella. The highest rate of larval survival (0.88) and larval growth index (9.77) were obtained in larvae which fed on corn flour. The relative growth rate (9.17 ± 0.33 mg/mg/day) and the efficiency of conversion of ingested food (12.08 ± 1.11%) were the highest rate for fifth instar larvae which fed on corn flour. The highest protein concentration in midgut of larvae (63.77 ± 2.31 mg/ml) and consequentially, proteolytic activity including tryptic (0.0012 ± 0.00 Umg-1) and chymotryptic (0.543 ± 0.001 Umg-1) were observed in larvae that fed on corn flour. According to the results obtained, corn (Var. 704) was the most suitable cereal for laboratory rearing of E. kuehniella, resulting in the highest rate of biological and physiological parameters.
... Lepidopteran larvae consume more food and due to the presence of multiple isozymes of proteases in their midgut they digest and assimilate the ingested food more rapidly and thus grow much faster than mammals and birds, with high metabolic rate. Trypsins, chymotrypsins, cathepsin-B-like proteases, elastases, aminopeptidases, carboxypeptidases, and many serine proteases are dominant proteolytic enzymes in the larval midgut (Chougule et al., 2008;Patankar et al., 2001;Srinivasan et al., 2006;Tabatabaei et al., 2011). The primary digestive proteinases of many insect species are serine proteinases, predominantly trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) (Reeck, et al., 1999;Terra & Ferreira, 1994). ...
We characterized trypsin- and chymotrypsin-like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio-formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76- and 14.05-fold for trypsin and chymotrypsin, respectively. Michaelis–Menten constants (Km) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ∼21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ∼29 and 34 kDa in SDS-PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio-formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.
... The results of this study showed that the α-amylase enzyme was active over an alkaline pH range (pH 9-12). According to the results of Tabatabaei et al. (2011), the α-amylase enzyme from Table 3. Correlation coefficients (r) of some biological and physiological characteristics of S. cerealella fed on different wheat cultivars with inhibition of α-amylase, soluble starch content and hundred-wheat weight. Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) was active in the pH range of 7-11. ...
The life history and digestive α-amylase activity of the Angoumois grain moth,
Sitotroga cerealella
Olivier (Lepidoptera: Gelechiidae) were studied on six wheat cultivars (Arg, Bam, Nai 60, Pishtaz, Sepahan and Shanghai) at 25 ± 1°C, relative humidity of 65 ± 5% and a photoperiod of 16:8 (L:D) h. A delay in the developmental time of
S. cerealella
immature stages was detected when larvae were fed on cultivar Sepahan. The maximum survival rate of immature stages was seen on cultivar Bam (93.33 ± 2.10%), and the minimum rates were on cultivars Nai 60 (54.66 ± 2.49%) and Sepahan (49.33 ± 4.52%). The highest realized fecundity and fertility were recorded for females which came from larvae fed on cultivar Bam (93.30 ± 2.10 eggs/female and 91.90 ± 3.10%, respectively); and the lowest ones were observed for females which came from larvae fed on cultivar Sepahan (49.30 ± 4.50 eggs/female and 67.4 ± 11.1%, respectively). The heaviest male and female weights of
S. cerealella
were observed on cultivar Bam (2.97 ± 0.02 and 4.80 ± 0.01 mg, respectively). The highest amylolytic activity of the fourth instar was detected on cultivar Bam (0.89 ± 0.04 mg maltose min
−1
), which had the maximum mean hundred-wheat weight (5.92 ± 0.19 g). One α-amylase isozyme was detected in the midgut extracts from the fourth instar larvae fed on different wheat cultivars, and the highest intensity was found in larvae fed on cultivar Bam. Correlation analyses showed that very high correlations existed between the immature period, fecundity and fertility on one side and inhibition of α-amylase, soluble starch content and hundred-wheat weight on the other. According to the obtained results, cultivar Sepahan is an unfavorable host for the feeding and development of
S. cerealella
.
... Their use in the insect control is notable based on several studies indicating their effectiveness in disruption of protease digestion. The lepidopteran larvae use a complex proteolytic enzymes including trypsins, chymotrypsins, elastases, cathepsin-B like proteases, aminopeptidases, and carboxypeptidases for protein digestion and many serine proteases are dominant in the larval gut (Patankar et al. 2001;Srinivasan et al. 2006;Chougule et al. 2008;Tabatabaei et al. 2011). Insect digestive proteases have different biochemical properties. ...
The biochemical properties of proteases from the digestive system of the fig tree skeletonizer moth, Choreutis nemorana, were determined. Gut extracts of C. nemorana larvae were analysed using different specific peptide substrates and proteinase inhibitors. The optimal pH and temperature for proteolytic activities using azocasein as substrate were obtained as pH 11 and 45°C, respectively. In the case of N-benzoyl-l-arg-p-nitroanilide as substrate, the enzyme showed the maximum tryptic activity at pH 11. The kinetic parameters of trypsin-like proteases indicated that the Km and Vmax values of trypsin in the gut of C. nemorana were 0.157 ± 0.006mM and 0.188 ± 0.005 μmol/min/mg protein. Using specific proteolytic inhibitors, the inhibitors including phenyl methane sulfonyl fluoride, N-p-tosyl-l-lys chloromethyl ketone and ethylene diamine tetraacetic acid showed the greatest inhibitory effect on total proteolytic activity. These results indicated that serine proteinases accounted for the major proteases in the gut of C. nemorana. Inhibition assays and zymogram analysis showed that only small amounts of cysteine proteases are present in the digestive system of C. nemorana.
... Glucosidase and galactosidase activities were determined against the substrates p-nitrophenyl-α-D-glucopyranoside (pNPαGlu, 5 mM, for α-glucosidase), p-nitrophenylβ-D-glucopyranoside (pNPβGlu, 5 mM, for β-glucosidase), p-nitrophenyl-α-Dgalactopyranoside (pNPαGal, 5 mM, for α-galactosidase), and p-nitrophenyl-β-Dgalactopyranoside (pNPβGal, 5 mM, for β-galactosidase) according to Razavi Tabatabaei et al. (2011). Reaction mixtures included 80 μl citrate-phosphate buffer (40 mM, pH 6), 10 μl enzyme extract, and 5 μl substrate, which were incubated at 35 • C for 30 min. ...
... The activity of α-glucosidase and β-glucosidase was detected according to Ramzi and Hosseininaveh (2010) and Tabatabaei et al. (2011) using p-nitrophenyl-α-D--glucopyranoside (pNaG, 5 mM), and p-nitrophenyl-β--D-glucopyranoside (pNbG, 5 mM) as the substrates, respectively. The assay was carried out by incubating 10 µl of the enzyme extract with 85 µl of 40 mM citrate-phosphate-borate buffer (pH 3 to 11), and 5 µl of the substrates for 30 min. ...
The Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) is a destructive polyphagous pest of many stored products. To interfere with the physiological processes, especially digestion, of the larval pest, more information on the regulatory mechanisms is needed. The brain extract from 1-day-old last instar larvae of P. interpunctellawas examined. In the bioassays, the midguts were treated with the brain extract, and the carbohydrase and protease activities were measured. The brain extract showed
increasing dose-dependent effects on α-amylase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, and trypsin secretion
in the larval midgut. The extract was further characterised and partially purified using high performance liquid chromatography
(HPLC). Several peptides were determined in the brain extract regulating hydrolase activities in the larval midgut of the pest.
... The assays were according to Razavi-Tabatabaei et al. (2011) by incubating 10 μl of the enzyme extract with 85 μl of 50 mM universal buffer (pH 3.0 -10.0) and 5 μl of each p-nitrophenyl-α-D-glucopyranoside (p NPαGlu, 5 mM) for α-glucosidase, or p-nitrophenyl-β-D-glucopyranoside (p NPβGlu, 5 m M) for β-glucosidase at 35°C for 30 min. Released p-nitrophenols were determined by measuring the absorbance at 405 nm after the addition of NaOH (2 M) to the reaction mixture. ...
Pistachio fruit hull borer, Arimania komaroffi Ragonot (Lep.: Pyralidae), is one the most important pests of pistachio in Iran. The larvae spin web as well as bore into young fruits, and the infested fruits fall off the trees. The second-generation adult moths appear in August and September, and their offspring feed on the fruit hull. Results indicated the presence of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and some proteases in the digestive tract of the pest. Highest activities of α-amylase, α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase were at pH 10, 7, 7, 6 and pH 6, respectively. Highest activities of trypsin, chymotrypsin and elastase of larval midgut were at pH 11. Zymogram analysis of α-amylase, α-glucosidase, β-glucosidase, tryptic, chymotryptic and elastase using native-PAGE revealed 1, 1, 2, 3, 3 and 2 bands of activity respectively, in A. komaroffi. One band was disappeared in the presence of the inhibitor TLCK, but no further inhibition by the inhibitors TPCK was observed. The results can be of help for designing new strategies for controlling the pistachio fruit hull borer based on natural proteases and carbohydrase inhibitors.
... Glucosidase and galactosidase activities were determined against the substrates p-nitrophenyl-α-D-glucopyranoside (pNPαGlu, 5 mM, for α-glucosidase), p-nitrophenylβ-D-glucopyranoside (pNPβGlu, 5 mM, for β-glucosidase), p-nitrophenyl-α-Dgalactopyranoside (pNPαGal, 5 mM, for α-galactosidase), and p-nitrophenyl-β-Dgalactopyranoside (pNPβGal, 5 mM, for β-galactosidase) according to Razavi Tabatabaei et al. (2011). Reaction mixtures included 80 μl citrate-phosphate buffer (40 mM, pH 6), 10 μl enzyme extract, and 5 μl substrate, which were incubated at 35 • C for 30 min. ...
The spined soldier bug, Podisus maculiventris, is a generalist predator of insects and has been used in biological control. However, information on the digestion of food in this insect is lacking. Therefore, we have studied the digestive system in P. maculiventris, and further characterized carbohydrases in the digestive tract. The midgut of all developmental stages was composed of anterior, median, and posterior regions. The volumes of the anterior midgut decreased and the median midgut increased in older instars and adults, suggesting a more important role of the median midgut in food digestion. However, carbohydrase activities were predominant in the anterior midgut. In comparing the specific activity of carbohydrases, α-amylase activity was more in the salivary glands (with two distinct activity bands in zymograms), and glucosidase and galactosidase activities were more in the midgut. Salivary α-amylases were detected in the prey hemolymph, demonstrating the role of these enzymes in extra-oral digestion. However, the catalytic efficiency of midgut α-amylase activity was approximately twofold more than that of the salivary gland enzymes, and was more efficient in digesting soluble starch than glycogen. Midgut α-amylases were developmentally regulated, as one isoform was found in first instar compared to three isoforms in fifth instar nymphs. Starvation significantly affected carbohydrase activities in the midgut, and acarbose inhibited α-amylases from both the salivary glands and midgut in vitro and in vivo. The structural diversity and developmental regulation of carbohydrases in the digestive system of P. maculiventris demonstrate the importance of these enzymes in extra-oral and intra-tract digestion, and may explain the capability of the hemipteran to utilize diverse food sources.
... The assays were according to Tabatabaei et al. (2011) by incubating 10 μl of the enzyme extract with 85 μl of 50 mM universal buffer (pH 3.0 -10.0) and 5 μl of each p-nitrophenyl-α-D-glucopyranoside (p NPαGlu, 5 mM) for α-glucosidase, or p-nitrophenyl-β-D-glucopyranoside (p NPβGlu, 5 m M) for β-glucosidase at 35 °C for 30 min. Released p-nitrophenols were determined by measuring the absorbance at 405 nm after the addition of NaOH (2 M) to the reaction mixture. ...
.The fig leaf roller or Fig-tree Skeletoniser, Choreutis nemorana (Lep.: Choreutidae), is a destructive pest of fig trees found in some fig-growing areas of Iran. The larvae feed on the upper level of leaves, near the main vein. In this study, digestive carbohydrases including α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase and proteinases including trypsin, chymotrypsin and elastase were investigated. The results showed that the carbohydrases were present in the alimentary tracts of the pest. Optimum pH for α-glucosidase and β-glucosidase activity was at pH 6.0 and 7.0, respectively. Maximum activity of α-galactosidase and β-galactosidase occurred at pH 6.0. Total proteolitic activity against the substrate azocasein was optimally occurred at pH 10.0. The greatest activity of trypsin, chymotrypsin and elastase was determined at pH 10.0, 11.0 and 11.0, respectively. Zymogram analyses using nitrocellulose membrane revealed two trypsin isoforms in which one of them was completely inhibited by Soybean Kunitz inhibitor and the other was notably inhibited.
The production of the black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae), has potential to promote sustainable food production, and is environmentally friendly through organic waste upcycling. Objective of this study was to determine the digestive enzyme activities and chemical composition of BSF larvae of 6, 12, and 18 days old at various substrate pH and temperature conditions during ontogenic development. Cellulase and chymotrypsin‐like protease (C) activities declined slightly with age, whereas a sharp increase in lipase activity was found with age. High levels of amylase, trypsin‐like protease (T), trehalase, and T/C ratio were present in 12‐day‐old BSF, with a gradual decrease in 18‐day‐old BSF. The highest crude protein and lipid levels were observed at 6 and 18 days of age, respectively. In conclusion, the activities of the various types of enzymes illustrate the polyphagous nature of BSF. Most of the digestive enzymes had high activity at an early age and then dropped at the end of the larval stage; however, the opposite trend was observed in lipase activity, with a high level of fat accumulation in the last stage of larval development.
Mulberry leaves (Morus alba var. Buriram 60) at the positions 1, 2, 3, 4 and 5 were harvested from 2nd, 3rd, 4th, 5th–6th and 7th–8th below the primordia, respectively; to evaluate on in vitro digestibility of carbohydrate (IVCD) and protein (IVPD) by using the crude enzyme extract from whole body of 3rd to 5th instar larva of mulberry silkworm (Bombyx mori). The crude enzymes were extracted from the whole body of larva against the reviewed data of gut extract from the previous studies. The optimal temperature and pH was similar between whole body and gut extracts, indicating the possible use of whole body for in vitro digestibility study. There was no statistical interaction between leaf position and developmental stage of larva. In all stages of larva, the leaf positions 2 and 3 were higher in IVCD than in the other positions (p < 0.05), whereas the lowest IVPD was found in position 1 (p < 0.05). The IVCD was highest in 3rd instar larva (p < 0.001) while the decrease trend of IVPD was observed in 4th, 3rd and 5th stages (p < 0.001), respectively. Based on the digestibility values, the preferred leaf positions for the mulberry silkworm instar larvae (3rd–5th) were leaf positions 2 and 3. This in vitro screening of the leaf supports the development of an artificial mulberry leaf-based diet for B. mori used in sericulture. © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society
The mid gut in insects is usually lined with a peritrophic membrane (PM), which protects it from microbial infections and physical contact with ingested food. Calcofluor is an optical brightener that has strong chitin-binding properties, which disrupt the chitin-protein structure of PM. In this study, we investigated the effects of calcofluor on the structure of the PM, activity of some common digestive enzymes and biological characteristics of the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). Twenty hours after feeding on a calcofluor-containing diet, the PM was completely eliminated. We recorded significant effects of calcofluor on the activity of digestive enzymes in the mid and hindguts of larvae. In both these regions of the gut, in particular, there were significant increases in the activity of α-amylase, α-galactosidase and trypsin. The activities of α-glucosidase and β-glucosidase in the midgut of treated larvae were lower than in the control, but were significantly higher in the hindgut of treated larvae. The larvae that fed on on calcofluor for 20 h, took longer to complete their larval and pupal stages, were lighter in weight and fewer reached maturity and laid fewer eggs. Results of this and previous studies highlight the crucial role of PM in enzyme recycling and digestion. PM disruption may negatively affect insect development on the one hand, by decreasing the efficiency of food digestion and, on the other, by increasing the costs associated with an increased enzyme demand and disrupted enzyme recycling mechanism.
Allothrombium pulvinum is a predatory mite capable of controlling aphid pests in agricultural and natural ecosystems. In this study, proteinase, carbohydrase and lipase activities were determined in the mite's gut using general and specific substrates, inhibitors and activators. The adult mites were dissected and their guts were removed and used as the enzyme source after homogenization and centrifugation. Gut pH of 5.3 was obtained in adults using pH dye indicators. Gut extracts showed an optimum proteolysis activity against haemoglobin under acidic condition (pH 3). The most effective inhibitor of proteinase activity was the cysteine proteinase inhibitor, E-64 [l-trans-leucylamido-(4-guanidino)-butane]. The optimum pH for acidic proteinase activity; the inhibition of proteinase activity by E-64; and increased proteinase activity in the presence of the activator l-cysteine revealed the presence of acidic proteinases, specifically cysteine proteinases, in the mite gut. Proteinase zymogram analysis showed at least three bands of proteinase activity in the gut extract. α-Amylase was optimally active against starch substrate at pH 5. The maximum activity of glucosidases (α and β) and galactosidases (α and β) occurred at pH 5 and pH 4, respectively. Zymogram analysis revealed at least two bands of activity for α-glucosidase and one band of activity for β-glucosidase. Lipase activity was also determined at pH 2–8 and the activity increased under alkaline conditions.
The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of alpha- and beta-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of alpha- and beta-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 mu mol(-1) min(-1) mg protein(-1), respectively. Activity of alpha- and beta-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of alpha- and beta-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 mu mol(-1) min(-1) mg protein(-1), respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 mu mol(-1) min(-1) mg protein(-1), respectively. Results showed that the optimal pH for alpha- and beta-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for alpha- and beta-glucosidase activity in the midgut was obtained as 45 degrees C. The addition of CaCl(2) (40 mM) decreased midgut beta-glucosidase activity whereas alpha-glucosidase activity was significantly increased at this concentration. The alpha-glucosidase activity, in contrast to beta-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive beta-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.
A beta-glucosidase was purified from the digestive fluid of the palm weevil Rhynchophorus palmarum L. (Coleoptera: Curculionidae) by chromatography on anion-exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gels, beta-glucosidase is a monomeric protein with a molecular weight of 58 kDa based on its mobility in SDS-PAGE and 60 kDa based on gel filtration. Maximal beta-glucosidase activity occurred at 55 degrees C and pH 5.0. The purified beta-glucosidase was stable at 37 degrees C and its pH stability was in the range of 5.0-6.0. The enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucoside, cellobiose, cellodextrins and required strictly beta-gluco configuration for activity. It cleaved glucose-glucose beta-(1-4) linkages better than beta-(1-2), beta-(1-3) and beta-(1-6) linkages. The catalytic efficiency (K(cat)/K(M)) values for p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 240.48 mM(-1)s(-1) and 134.80 mM(-1)s(-1). Beta-glucosidase was capable of catalysing transglucosylation reactions. The yield of glucosylation of 2-phenylethanol (20 %), catalysed by the beta-glucosidase in the presence of cellobiose as glucosyl donor, is lower than those reported previously with conventional sources of beta-glucosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transglucosylation reactions (pH 6.6).
A combination of ion-exchange chromatography, preparative electrophoresis and gel filtration chromatography allowed a 1209-fold purification of one of the two major digestive alpha-amylases from larvae of the larger grain borer, Prostephanus truncatus Horn. The purified enzyme showed a molecular mass of 60.2 kDa, an isoelectric point of 4.7 and an optimal pH for activity of 6.0. The enzyme was heat labile and it was recognized by proteinaceous inhibitors from amaranth seeds (Amaranthus hypochondriacus), whereas extracts from maize (Zea mays) and tepary bean (Phaseolus acutifolius) produced very low inhibition. When the enzyme was measured at different stages of development, maximal activity was found in the second instar larvae. Activity drastically decreased to a very low level during the pupae stage and increased again at the adult stage. A zymogram of the different developmental stages showed two main bands of alpha-amylase activity, which almost disappeared at the pupae stage to increase again during the adult stage, revealing a new, smaller band. This new band may be required for a better adaptation of the adult insect to its new environment.
The nutritional value of various starches, incorporated in marginal diets, for larval development of the flour beetle, Tribolium castaneum (Herbst), was assayed at 32°. Wheat, maize and rice starches were optimal. Larvae died when fed raw potato starch at 32°, but survived and developed slowly at 28°. Development was slightly better with boiled potato starch. The β-amylase limit dextrin “core” of potato starch — containing much esterified phosphorus — was devoid of nutritional value, in contrast with the high nutritional value of β-amylase limit dextrin of commercial amylopectin. The amylolysis of starch by Tribolium larvae is attributed mainly to the activity of α-amylase, the characteristics of which were examined. The in vitro hydrolysis of starches is loosely correlated with their nutritional value: amylopectin and β-amylase limit dextrin were well digested, but non-solubilized amylose, only slightly. Boiled potato starch was well digested, whereas raw potato starch was not. The similarity of these in vivo and in vitro results to those obtained with higher animals is noted, and the usefulness of this observation for the bioassay of starches is suggested.
The pH of the midgut digestive juices of two species of carnivorous lepidopterous larvae and of 40 species of adult Lepidoptera belonging to 16 families was determined. In all species it was alkaline. From these records, and from those available from the literature for phytophagous, wool-eating, and wax-eating lepidopterous larvae, it is possible to generalize that midgut alkalinity is characteristic of the order Lepidoptera and that it is not dependent upon feeding habits.
The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. Vmax was higher and Km was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30°C to 35°C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl2, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl2, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.
The midgut of Dysdercus peruvianus is divided into three main sections (V1-V3) and is linked through V4 to the hindgut. The distribution of α-galactosidase activity in the different gut segments of D. peruvianus females was studied. α-galactosidase hydrolyzes the trisaccharide raffinose, the major carbohydrate of cotton seeds, on which the insects live. In D. peruvianus midgut, α-galactosidase activity is mainly found in soluble fractions of V1 contents. However, a comparison between specific activities using different α-galactosidase substrates in cotton seed extracts, V1 tissue homogenate, and midgut contents suggested that the contribution of the enzymes from seeds may be very significant. Gel filtration on Sephacryl S-200 of samples from seed extracts, V1 tissue, and V1 contents revealed that in all samples raffinose hydrolysis is accomplished by α-galactosidases with similar Mr (30,000 ± 3,000) and does not involve the activity of a β-fructosidase. Thermal inactivation studies of extracts from the three sources suggested that there was only one molecular form of the insect α-galactosidase and that the activity found in V1 contents includes enzymes derived from the seed kernel. In insects fed with cotton seeds, the α-galactosidase activity increased in parallel with diet ingestion. In starved insects fed with tablets of sucrose plus raffinose, an increase in α-galactosidase activity was also observed, confirming that the insect is able to synthesize part of the gut enzyme. The results indicated that raffinose digestion starts in V1 utilizing α-galactosidases derived from the seed kernel and by an additional α-galactosidase synthesized by insect tissues. The action of α-galactosidases liberates galactose and sucrose, which are sequentially hydrolyzed by the major membrane-bound α-galactosidase releasing glucose and fructose in V1 and V2 lumina. Arch. Insect Biochem. Physiol. 34:443–460, 1997. © 1997 Wiley-Liss, Inc.
In Abracris flavolineata midguts, cellulose is hydrolyzed by at least three enzymes, whereas the most active hemicellulases are a laminarinase (pH optimum 5.7, Mr 146 k) and three lichenases (pH optima in the range 5–7.3; Mr values: 22, 71, and 97 k). Digestion of hemicellulose is completed by β-glucosidases described elsewhere and by an α-mannosidase (pH optimum 4.7, Km 1.7 mM). There are a major and two minor amylases (pH optimum 6.5; Mr values: 42, 45, and 108 k) activated by chloride. Two of the α-glucosidases (Mr 74 and 94 k) are active on maltose and hence should finish the digestion of starch. It is not clear what is the natural substrate of the remaining α-glucosidase (Mr 93 k). The major α-galactosidase (Mr 112 k) is active on melibiose and raffinose, whereas the minor (Mr 70k) may be active on digalactosyl diglycerides. The effect of pH on azocasein hydrolysis and electrophoresis data suggest that trypsin is a major and chymotrypsin and cysteine proteinase are minor enzymes. The cysteine proteinase may derive from the leaves ingested by the grasshopper, taking into account its activity in leaves. Protein digestion is finished by two soluble aminopeptidases (Mr 92 and 105 k), a major membrane-bound aminopeptidase (Mr 97 k) and two membrane-bound dipeptidases (Mr 87 k). The sizes of the digestive enzymes recovered in A. flavolineata crops suggest that the pores of the semi-fluid caecal peritrophic membrane have diameters larger than 8.6 nm.
Serine protease activity has been extracted and partially purified from the alimentary tract of larval Helicoverpa armigera. The major activity was present in the midgut contents. Characterisation of this protease, using BApNA as substrate, gave a pH optimum of pH 9.5–10. Km and Vmax were 0.254 ± 0.032 mM and 0.351 ± 0.037 μmol pNA produced min−1 mg protein−1 respectively. Inhibition was effected by TLCK but not TPCK; this together with the failure to hydrolyse BTpNA or SUPHEPA, indicated trypsin-like but not chymotrypsin-like specificity. Comparison between the insect protease and bovine trysin revealed differences in inhibitor sensitivity; the insect protease being unaffected by OMTI, whilst showing greater inhibition by chymostatin and SBTI. The kinetics of the interactions between the insect protease activity and various plant-derived protease inhibitors were determined. Unlike bovine trypsin, the insect enzyme was not affected by calcium ions or the divalent chelating agent, EDTA. Partial purification by ion-exchange chromatography, followed by SDS-PAGE, showed that protease activity was largely associated with a polypeptide of .
The Khapra beetle, Trogoderma granarium is a serious post-harvest pest of wheat. Digestive proteinase and amylase activities of larvae and adult insects were investigated using general and specific substrates and inhibitors. Gut pH in larvae was determined to be slightly acidic in the midgut and more acidic in the hindgut. However, midgut extracts from larvae showed an optimum activity for proteolysis against azocasein and haemoglobin at alkaline pH (broad optimum in the range 8–11). Proteinase extracted from larval guts hydrolyzed the synthetic substrates BApNA(Nα–benzoyl–L–arginine–p–nitroanilide) and SAAPPpNA (N–succinyl–alanine–alanine–proline–phenylalanine–p–nitroanilide) for trypsin- and chymotrypsin-like activities, respectively. The specific substrate for elastase-like activity SAAApNA (N–succinyl–alanine–alanine–alanine–p–nitroanilide) was not hydrolyzed by midgut extract of larvae. The most effective inhibitors of azocaseinolytic hydrolysis were trypsin and chymotrypsin-like serine proteinase inhibitors. Casein zymogram analysis revealed at least six proteinase bands in the larval midgut extract and showed the presence of trypsin-like serine proteinases. No cysteine proteinases were detected in larval midgut extract using specific cysteine proteinase substrates, inhibitors and activators. Carboxypeptidase A activity also was detected in larval midgut extract using FAPP(N-(3-(2-furyl) acryloyl)-l-phenylalanyl-l-phenylalanine) and showed optimal activity at alkaline pH. Activity of α-amylase using starch as substrate, was determined to be maximal around neutral pH (pH 6–8), which is correlated with the pH prevailing in the midgut. No proteolytic and amylolytic activities were detected using conventional substrates in the digestive system of the adult suggesting no significant protein and carbohydrate digestion occurs in the adult beetle which does not need to feed.
Ectomyelois ceratoniae (Zeller),a carob moth, is a date pest that was recently introduced into the Coachella Valley of California. Infestation of the current season's crop of 'Deglet Noor' dates by this moth or by nitidulid beetles does not occur in the khalal stage of development of date fruits unless summer rains occur. Rain and accompanying high relative humidity results in fruit cracks, fungus infection, and susceptibility to insect infestation of khalal stage dates. In summers without rain, significant nitidulid populations fail to develop. In dry summers, E. ceratoniae oviposits on fallen dates of the previous season that are lodged in frond axils. They begin to oviposit on the new crop when the rutab or ripening stage begins. The appearance of the rutab stage is forecast by the development of a glossy finish on khalal dates and is the time when the first insecticide treatment should be applied. Commercial treatments are applied exclusively by powerful dusting equipment that delivers a dust stream through a hand-directed tube. Only insecticides of low toxicity to the operator can be used. During three seasons, trials were made of various application schedules of a malathion (5%)dust. In a summer with rain-induced nitidulid populations, four applications at 2-wk intervals adequately controlled both beetles and moths. Treatment should begin just ahead of the development of the rutab stage of fruit development. In rainless summers, three treatments applied at 3-wk intervals successfully controlled E. ceratoniae. Early initiation of the treatment schedule did not improve control.
Levels of α-amylase activity between 0.6 and 5.0 µmoles maltose minute-1 gram insect-1 occurred in larval midgut extracts from 4 species of lepidopterous pests-light brown apple moth, Epiphyas postvittana (Walker), Planotortrix octo (Dugdale), and Ctenopseustis Obliquana (Walker) are pests of apple leaves and fruit, and potato tuber moth, Phthorimaea Operculella (Zeller), is a pest of potato tubers and foliage. Activity response curves described the optima for activity at a highly alkaline pH range (pH 10.5 in leafrollers and pH 9.5 in potato tuber moth). This result is consistent with the known high midgut pH in other species of Lepidoptera and with the pH optima of their digestive proteases. The 3 wheat derived α-amylase inhibitors inhibited enzyme activities of all 4 species of Lepidoptera, but the other 3 (barley tetramer, Streptomljces, and kidney bean inhibitors) had no effect. Wheat dimeric inhibitor (30 µM) completely inhibited α-amylase activity in potato tuber moth midgut extracts.
In the field in Iraq Ectomyelois ceratoniae had four generations on an artificial diet and four to five generations on pomegranate fruits. On an artificial diet the mean time for development from egg to adult ranged from 41 to 59 days for the first three generations. Fourth-generation larvae entered diapause in the fall and averaged 241 days. Insect population increased progressively during the fruiting season. Mean percentage of infestation increased from 20% by early generations to more than 80%. High infestation was observed in southcentral areas of Iraq. Orchards in which sanitation was poor were highly infested.
Properties of amylases in homogenates of midguts of larvae of the granary weevil, Sitophilus granarius (L.), and maize weevil, Sitophilus zeamais Motchulsky, were studied. Amylases from both species were active in mildly acid buffers. The pH optima were 4.75 for S. zeamais and 5.0 for S. granarius. Amylases from both weevils were activated by Cl− and stabilized against thermal inactivation by Ca2+. Each species hydrolyzed soluble starch and amylopectin with identical kinetic parameters. However, different values for soluble starch were obtained in each species: 0.077% for S. granarius and 0.13% for S. zeamais. Action patterns of amylases of S. granarius and S. zeamais were identical and were similar to that of porcine pancreatic α-amylase. The larval enzymes were of the endoamylase type and should be classified as α-amylases (E.C. 3.2.1.1). NaHCO3 extracts of wheat strongly inhibited the amylases from both weevils. Extracts equivalent to 50 μg flour inhibited about 80% of the amylase activity in each species. Extract concentrations equivalent to 400 μg flour resulted in almost complete inhibition. Two strongly anionic amylase isozymes were demonstrated in larval midgut homogenates from S. zeamais by vertical slab polyacrylamide gel electrophoresis. A single anionic amylase was found in S. granarius.
Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were purified by using a sequential procedure of ammonium sulfate precipitation, glycogen-complex formation, and ion exchange chromatography. Amylase of S. oryaze was purified 47.4-fold to a specific activity of 478 units/mg protein. One amylase unit equals 1 mg maltose hydrate produced/min at 30°C. Amylase of S. granarius was purified 85.4-fold to a specific activity of 453 units/mg protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a single protein with an isoelectric point of pH 3.76. Purified amylases of both species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had double energies of activation calculated from Arrhenius plots. Based on fresh weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae contained three-fold and eight-fold more amylase than S. granarius and S. zeamais Motschulsky, respectively. High amylase levels in S. oryzae may provide this species with an adaptive advantage when feeding on cereals containing naturally occurring amylase inhibitors.
Proteinase and amylase activities in larval midguts of the bruchid beetle Zabrotes subfasciatus (Boh.) (Coleoptera: Bruchidae) reared on cowpea (Vigna unguiculata (L.) Walp.) seeds were investigated. We could detect and isolate a proteolytic activity with a pH optimum of 5.5 (on azo-casein as substrate) which was activated by thiol reagents and inhibited by several compounds reactive against -SH groups. None of the plant protein inhibitors of serine proteinases utilized were effective inhibitors of this activity. This activity has characteristics of a cysteine class proteinase. We could also detect and isolate a proteolytic activity with a pH optimum of 3.5 (on hemoglobin as substrate) which was not influenced by activators or inhibitors of cysteine, serine, or metalloproteinases. This activity was totally inhibited by pepstatin, a specific inhibitor of aspartic proteinases. We conclude that this activity is due to an aspartic class proteinase. We found also that the aspartic class proteolytic activity is higher than the cysteine class proteinase activity in the midguts of Z. subfasciatus. This seems to be contrary to what is found in Callosobruchus maculatus (F.) larvae midguts. An amylolytic activity with the charateristics of an α-amylase was also detected and isolated.
Field observations and recording of carob moth [Spectrobates ceratonia (Zell.)] infestation in almonds revealed the following pattern of generation sequence. The moths overwinter as larvae in
nuts left on the trees after harvest; these pupate in mid-February and adults emerge from mid-March until June, with a peak
emergence in April-May. Emerging females lay mainly on new nuts infected by the anthracnose disease as well as on old nuts,
if available. The first summer generation reached adulthood from May to July, at which time hullsplit nuts of the new crops
are available for oviposition. One to two generations develop on the hullsplit nuts before winter, thus bringing the annual
average to three or four generations. Eggs are dispersed evenly among the nuts, usually with a single egg laid on each nut.
The hatching larvae feed on the kernel, causing economic damage, or on the hull. Damage may be reduced by early harvesting.
Natural parasitism of the carob moth on almonds was insignificant. A few other moth species infest almond nuts, but do not
cause any significant damage.
The pH of the gut was determined in vitro in six species of termite by means of indicator dyes and a pH electrode. In the lower termite Zootermopsis nevadensis the pH was close to neutrality throughout, ranging 6.0–7.5, but in Reticulitermes lucifugus, acid conditions (pH 5.5–6.0) occurred in the crop and paunch. In the higher termites Nasutitermes costalis, Microcerotermes arboreus, Cubitermes severus and Procubitermes aburiensis, there was a common trend of incresing pH from the crop, which was slightly or moderately acidic, to the first proctodaeal segment (P1) where moderately (N. costalis) and strongly (M. arboreus, C. severus and P. aburiensis) alkaline conditions prevailed. A pH of 10.4 was measured in C. severus, equalling the highest recorded in any insect. In the posterior regions of the hindgut there was a return towards neutral or acidic conditions. When termite guts were homogenized with air-saturated Ringer's solution, the dissolved O2 content of the Ringer's was reduced. This was shown to be largely attributable to an oxygen deficit generated within the gut in situ. The combined effects of strongly alkaline conditions and reduced oxygen tension on digestive processes and intestinal micro-organisms are discussed.
Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.
Salivary and intestinal proteolytic enzymes were studied in the predaceous heteropteran, Zelus renardii. Proteinases from Z. renardii's salivary gland complex (SGC), anterior midgut (AMG) and posterior midgut (PMG) were studied with hemoglobin, BApNA, BTNA, HPLA, HA and LPNA used as substrates. The SGC was shown to be an important source of general proteinase and endopeptidase activity, but exopeptidase activity (LPNA, HPLA and HA hydrolysis) was limited to the AMG and PMG. The AMG was also a source of considerable endopeptidase activity. Hemoglobin or BApNA hydrolysis occurred at a broad range of temperatures and pH with most activity observed at 35–45°C and at pH 7.0–8.5. The pH of the prey increased, as it was being pre-orally digested. The salivary trypsin-like enzyme had a molecular weight of approx. 27 kDa and eluted as two major activity peaks from an ion exchange column. BApNA hydrolysis was inhibited by soy proteinase inhibitor. Digestion, with respect to protein hydrolysis, is organized into an orderly and highly efficient process, differing greatly from that of hematophagous reduviids, beginning with pre-oral digestion of the prey's internal structures whose liquefaction permits ingestion and further digestive processing of prey within the predator's gut.
Endoproteases from the lumen of the midgut of larvae of Spodoptera littoralis were characterised with respect to their substrate specificity and pH optimum. Alkaline serine protease activities corresponding to trypsin and chymotrypsin were detected. Cysteine, aspartic or metalloprotease activities were not found, nor was serine protease activity associated with microbes. Chymotrypsin hydrolysed SA2PPpNA, SA2PLpNA, BTEE, SA3pNA and casein, but not BTpNA, ALpNA or SPpNA, indicating an extended binding site. It was effectively inhibited by chymostatin, PMSF, antipain, SBTI, BBTI, LBTI, CEOI and TEW, but unaffected by TPCK. Trypsin hydrolysed BApNA, TGPLpNA, TAME and BAEE. It was effectively inhibited by TLCK, benzamidine, leupeptin, chymostatin, antipain, CEOI, aprotinin, pCMPS and E-64. Chymostatin, antipain and CEOI were the only three inhibitors which were effective against both endopeptidases. Trypsin and chymotrypsin have a molecular weight of 24,000 and 19,000, respectively, as determined by gel filtration. The optimal pH for both enzymes was pH 10.0 using p-nitroanilide substrates, but lower for esterase substrates (between pH 8.0 and 9.5) and higher for casein hydrolysis by chymotrypsin (pH 11.0).
Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or l-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM l-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe–Phe–CHN2 and Z-Phe–Ala–CHN2, suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.
Different amylase enzymes were identified by analysis of digestive fluid and haemolymph in diapausing and nondiapausing strains of silkworm, Bombyx mori. The diapausing strain showed negligible digestive amylase activity at a pH range of 3–11, while the nondiapausing strain registered strikingly higher amylase activity at pH 9.2. Higher levels of undigested starch was found in the faecal matter of the diapausing strain, which is consistent with the negligible digestive amylase activity. Development specific expression of haemolymph amylase activity was seen in nondiapausing and diapausing strains. In the nondiapausing strain the digestive amylase activity was at its peak during intermoult and depressed during moult. PAGE analysis revealed the occurrence of only anodal digestive and haemolymph amylases in the diapausing strain, whereas both cathodal and anodal enzymes were seen in the digestive fluid and haemolymph of the nondiapausing strain.
Plants and insects have been co-evolving for at least 300 million years; during this time plant species have been under continuous selection pressure from herbivorous insect predators, and, in the absence of the ability to avoid predators by movement, have been forced to rely on physical and chemical defensive mechanisms. Physical mechanisms include the presence of hairs on the surfaces of plant tissues, spines, exuded gums and toughened surface layers, such as hardened seed coats. Chemical defensive mechanisms include all the cases where a plant metabolite present in the plant tissues is toxic to predators, or interferes with the normal growth and development of predators, or deters predators by its taste or smell (Norris and Kogan, 1980).
A Helicoverpa armigera larval midgut cDNA library from larvae raised on an artificial, protein-rich, inhibitor-free diet contained very large numbers of serine proteinase positive clones. DNA sequencing of six random positive cDNAs and 12 PCR derived products identified trypsin genes classifiable into three families, and chymotrypsin and elastase genes classifiable into a single family each. Genomic blots established that the most highly expressed of the trypsin families contained about 18 genes, and that the chymotrypsin and elastase families contained about 14 and 2 genes respectively. The levels of mRNA corresponding to the highly expressed trypsin and chymotrypsin families were determined following chronic ingestion of four proteinase inhibitors. Compared to insects on an inhibitor-free diet, chymotrypsin mRNA was increased by all inhibitors, and trypsin mRNA levels decreased. This occurred independent of whether the inhibitor was solely a trypsin inhibitor (aprotinin), an inhibitor of both trypsin and chymotrypsin (proteinase inhibitor II, soybean trypsin inhibitor) or predominantly a chymotrypsin inhibitor (proteinase inhibitor I). Changing the protein level of the diet did not affect trypsin mRNA levels, but chymotrypsin mRNA levels decreased with increasing dietary protein.
Thesis (Ph. D.)--University of California, Riverside, 1988. Includes bibliographical references.
C. chinensis larval amylase is activated by Ca(2+) and inhibited by Cl(-) and EDTA (K(i) 6.7x10(-3)m). GSH and 2-mercaptoethanol activate, presumably at different sites, as 2-mercaptoethanol interferes with Ca(2+) activation, whereas GSH enhances it. The inhibition by iodoacetic acid and N-ethylmaleimide (K(i) 1.55x10(-2)m) suggest that free thiol groups are essential for activity. The pH optimum of 5.2-5.4 is moved to 5.6-5.8 by Ca(2+) and 2-mercaptoethanol. The activation energy is 7270 cal/mol, and is not affected by Ca(2+) and 2-mercaptoethanol. K(m) for soluble starch is 2.3mg/ml.
We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.
The adult coffee berry borer (Hypothenemus hampei Ferrari [Coleoptera: Scolytidae]), a major insect pest of coffee, has two major digestive alpha-amylases that can be separated by isoelectric focusing. The alpha-amylase activity has a broad pH optimum between 4.0 and 7.0. Using pH indicators, the pH of the midgut was determined to be between 4.5 and 5.2. At pH 5.0, the coffee berry borer alpha-amylase activity is inhibited substantially (80%) by relatively low levels of the amylase inhibitor (alphaAI-1) from the common bean, Phaseolus vulgaris L., and much less so by the amylase inhibitor from Amaranthus. We used an in-gel zymogram assay to demonstrate that seed extracts can be screened to find suitable inhibitors of amylases. The prospect of using the genes that encode these inhibitors to make coffee resistant to the coffee berry borer via genetic engineering is discussed.
Two beta-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl beta-D-glucoside (NPbetaGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl beta-D-galactoside (NPbetaGal) better than NPbetaGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0. The Mr 47,000 BG N-terminal sequence has high identity to plant beta-glycosidases and to mammalian lactase-phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase-phlorizin hydrolase.
There are three midgut alpha-galactosidases (TG1, TG2, TG3) from Tenebrio molitor larvae that are partially resolved by ion-exchange chromatography. The enzymes have approximately the same pH optimum (5.0), pl value (4.6) and Mr value (46000-49000) as determined by gel filtration or native electrophoresis run in polyacrylamide gels with different concentrations. Substrate specificities and functions were proposed for the major T. molitor midgut alpha-galactosidases (TG2 and TG3) based on chromatographic, carbodiimide inactivation, Tris inhibition, and on substrate competition data. Thus, TG2 would hydrolyse alpha-1,6-galactosaccharides, exemplified by raffinose, whereas TG3 would act on melibiose and apparently also on digalactosyldiglyceride, the most important compound in the thylacoid membranes of chloroplasts. Most galactoside digestion should occur in the lumen of the first two thirds of T. molitor larval midguts, since alpha-galactosidase activity predominates there. Spodoptera frugiperda larvae have three midgut alpha-galactosidases (SG1, SG2, SG3) partially resolved by ion-exchange chromatography. The enzymes have similar pH optimum (5.8), pl value (7.2) and Mr value (46000-52000), and at least the major alpha-galactosidase must have an active carboxyl group in the active site. Based on data similar to those described for T. molitor, SG1 and SG3 should hydrolyse melibiose and SG3 should digest raffinose and, perhaps, also digalactosyldiglyceride. The midgut distribution of alpha-galactosidase activity supports the proposal that alpha-galactosidase digestion occurs at the surface of anterior midgut cells in Spodoptera frugiperda larvae.
Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.
Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.
Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides. By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis. Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.
Plants and herbivores have been co-evolving for thousands of years, and as a result, plants have defence mechanisms that offer protection against many herbivores such as nematodes, insects, birds and mammals. Only when a herbivore has managed to adapt to these defence mechanisms does it have the potential to become a pest. One such method of plant defence involves the production of protease inhibitors (PIs). These inhibitors are proteins that may be found constitutively in various parts of the plant, or may be induced in response to herbivore attack. PIs work at the gut level, by inhibiting the digestion of plant protein. This review focuses on insect herbivores and looks at the mechanisms involved in the role and function of PIs in plant defense against insects, as well as at the ability of well adapted species to overcome the effects of these plant PIs.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.