Comparison of the effects of cereal and legume proteinaceous seed extracts on α-amylase activity and development of the Sunn pest

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is a significant limiting factor in the production of wheat and barley in many areas of the world. In the current study, the effect of semi-purified proteinaceous extracts of seeds on digestive enzymes, and the growth and development of the Sunn pest were studied. The results showed that the purified α-amylase inhibitor from Triticum aestivum (type І) and rice semi-purified seed extract did not significantly affect the Sunn pest α-amylase activity. However, bean and cowpea seed extracts significantly affected α-amylase activity in vitro. For example, the bean seed extract at concentrations of 0.125 and 2.0 mg · mL^− 1 inhibited α-amylase activity of the pest by 15% and 45%, respectively, while the cowpea seed extract, at the same concentrations, inhibited α-amylase activity of the pest by 9% and 40%, respectively. Further, incorporation of the seed extracts into the insect diet showed that the rice seed extract did not affect insect development time, while bean and cowpea seed extracts at high concentrations (e.g., 3.0%) significantly affected nymphal development time and survivability (P > 0.05). These results show that semi-purified seed extracts affect α-amylase activity, developmental time, and survivability but not the adult weight of the Sunn pest.     

Comparison of α- and β-mannosidase activity in the three cereal pests,Haplothrips tritici Kurdjumov (Thysanoptera: Phlaeothripidae), Rhopalosiphum padi L. (Homoptera: Aphididae) and Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

The Sunn pest, Eurygaster integriceps, the bird cherry-oat aphid, Rhopalosiphum padi, and wheat thrips, Haplothrips tritici are the major pests of wheat and other cereals in a wide area of the world. All these three insect species could produce damage to the wheat to some extent. Therefore, the purpose of the present study was to determine α- and β-mannosidase of the three mentioned insect pests. These insects were collected from the wheat farm and their guts (the Sunn pest and the aphid) and salivary glands of Sunn pest were removed. However, regarding tiny body of thrips, the whole body used in order to extract the enzymes. The enzymes, including α- and β-mannosidase activity, were measured by the hydrolysis of p-nitrophenyl-α-d-mannopyranoside (pNPαGal) and p-nitrophenyl-β-d-mannopyranoside (pNPβGal), respectively, using phosphate citrate buffer (pH 5.0). Mannosidases were not active in all three tested insect species, and also there were significant differences in activities of the two enzymes in three species. The greatest activity of α-mannosidases was observed in the Sunn pest salivary glands, E. integriceps, and the least activity was found in Sunn pest midgut with no activity. However, the activity of β-mannosidase was established in Sunn pest midgut, but there was no activity in the aphid midgut, R. padi. Activities of these two enzymes were modest in the thrips, H. tritici. The greatest amount of β-mannosidases in the Sunn pest midgut makes sense, since the Sunn pest is the main pest in the wheat farm that can feed on wheat grains. In the wheat grains, the highest amount of glycoproteins and glycolipids are present. Thus, it has been known that these enzymes (α- and β-mannosidases) are active on digestion of carbohydrates.     

Sunn pest,Eurygaster integriceps Putton (Hemiptera: Scutelleridae), digestive α-amylase, α-glucosidase and β-glucosidase

Morphology, pH and carbohydrate hydrolyzing enzyme activities of the Sunn pest gut were investigated in this study. The Sunn pest midgut is separated into the first ventriculus (V1), the second ventriculus (V2), the third ventriculus (V3) and the fourth ventriculus (V4). The first three regions of the midgut were acidic (pH 5.0–5.2), while the fourth region of the midgut and rectum was moderately acidic (pH 6.2–6.4 and pH 6.5–6.8, respectively). Activity of α-amylase was highest at pH 6 to 7, which correlates with the pH of the midgut. The optimum pH for α-glucosidase and β-glucosidase is 4 to 6 and 5 to 6, respectively. Different gut regions had different carbohydrate hydrolyzing enzyme activities. Carbohydrate hydrolyzing enzyme activities in V2 and V4 were the same, but activities in V1 were slightly higher than in V2 and V4 and lower than in V3. Levels of α- and β-glucosidase activities were similar in various midgut sections. However, the V3 had the highest activity followed by V4, V2, V1, respectively.     

Effect of plant a-amylase inhibitors on sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), alpha-amylase activity

Plant-insect interaction is a dynamic system, subjected to continual variation and change. In order to reduce insect attack, plants developed different defence mechanisms including chemical and physical barriers such as the induction of defensive proteins, volatiles that attract predators of the insect herbivores and secondary metabolites. Proteinaceous inhibitors of alpha-amylase and proteases are widely distributed in cereals, legumes and some other plants. Because of the possible importance of these inhibitors in plant physiology and animal nutrition, extensive research has been conducted on their properties and biological effects. Sunn pest like other insect pests of wheat lives on a polysaccharide-rich diet and depends to a large extent on effectiveness of their alpha-amylases for survival, a-amylase (1-4-alpha-D-glucan glucanohydrolase) hydrolyses starch, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic linkages and has a major role in the utilization of polysaccharides. The enzyme inhibitors act on key insect gut digestive hydrolyses, alpha-amylase. Several kinds of a-amylase inhibitors present in seeds and vegetative organs of plant, act to regulate number of phytophagous insects. Therefore, the aim of the current study is to study cereal proteinaceous inhibitors of insect digestive enzymes and their potential use as resistance factors against Sunn pest. The proteinaceous inhibitors from different cereal species including barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were extracted and tested in in vitro condition against Sunn pest alpha-amylase. Extraction was made with NaCl (0.15 M) at room temperature and further purification was done by ammonium sulphate precipitation. It was found that fractions obtained from barley had more inhibitory effect on amylase activity of Sunn pest than fractions obtained from wheat. Knowledge gained through these studies can be used to select resistant plant against insect pest.     

Anti-glycation effect and the α-amylase,lipase, and α-glycosidase inhibition properties of a polyphenolic fraction derived from citrus wastes

Abstract

The advanced glycation end products (AGEs) constitute a wide variety of substances synthesized from interactions between amino groups of proteins and reducing sugars, which excess induces pathogenesis of chronic diseases. Brazil is the major producer of citrus, a low-cost source of hesperidin, which is a polyphenol recognized for its capacity to inhibit AGEs formation. This is the first work to evaluate the effects of a polyphenolic fraction derived from citrus wastes on the antiglycation and on the inhibition properties of digestive enzymes on the possibility to process these wastes in high value-added products. At concentrations of 10, 15 and 20?mg/mL inhibition of AGEs was higher than 60%. The extracts were able to inhibit by 76% the activity of pancreatic lipase and by 98% the activity of α-glucosidase. For the α-amylase the inhibition capacity was lower than 50%. Strong correlation was obtained among anti-glycation with polyphenolic content and antioxidant capacity.     

Identification and biochemical characterisation of α-amylase in the alimentary tract of Mediterranean fruit fly,Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Mediterranean fruit fly (Medfly), Ceratitis capitata, is an important pest of many fruit crops in temperate and subtropical regions worldwide. α-Amylases are hydrolytic enzymes involved in carbohydrate metabolism in insects. There is no report about α-amylase activity in C. capitata in literature. So, the aim of the current study was biochemical characterisation of α-amylase in the alimentary canal of the pest to gain a better understanding of digestive physiology of the insect. α-Amylase of Medfly was extracted and characterised using starch as the substrate. The results showed the presence of α-amylase activity in the gut of the insect for carbohydrate digestion. Optimum activity of the enzyme occurs at pH 8.0 and 40?°C. The most effective activator of the enzyme was determined in treatment with 20?mM CaCl₂. Na⁺, K⁺ and Mg²⁺ ions also activated the enzyme. Native PAGE of α-amylase showed two isoenzymes suggesting the importance of α-amylase in the carbohydrate digestion in the insect. Understanding of the digestive physiology and α-amylase activity of Medfly is important when new management strategies for this economically important pest are devised.     

Biochemical characterisation of digestive proteases and carbohydrases of the pistachio fruit hull borer,Arimania komaroffi Ragonot (Lepidoptera: Pyralidae)

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.     

The effect of cereal seed extracts on amylase activity of the rose sawfly,Arge rosae Linnaeus (Hymenoptera: Argidae)

The sawfly, Arge rosae Linnaeus (Hymenoptera: Argidae) is a serious pest of rose plants in a vast area of the world including Iran. Pesticides are applied in order to control in areas where infestation is high. So, there is a need for more environmentally benign alternatives to control this insect pest in urban areas. Therefore, the aim of the current study was to study the effect of cereal seed proteinaceous extracts including wheat and triticale on the insect α-amylase. Wheat and triticale seed proteins as well as fourth instars larvae α-amylase were extracted. The results showed that there are two different α-amylase isoenzymes in the insect gut, one major band and the other minor band, which had optimal activity at alkaline pH (pH 8.0). The effect of five concentrations including 0.42, 0.21, 0.105, 0.05 and 0.025?mg?ml^?1 of each seed extract was tested on α-amylase activity of the larvae gut. At the highest concentration (0.42?mg?ml^?1), wheat seed extract caused 74% inhibition of the enzyme activity while triticale seed extract inhibited 62% enzyme activity. Experiments proved that seed proteinaceous extract affected the enzyme activity in a pH-dependant manner.     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

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. V_max was higher and K_m 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.     

A amylase activity of nymphal stages of sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae)

Wheat production in Iran has changed substantially over the past one or two decades with development of higher-yielding cultivars and improved methods of planting. Sunn pest, Eurygaster integriceps (Heteroptera: Pentatomidae), is the most important cereal pest in Iran. Sunn pest like other insect pests of wheat lives on a polysaccharide-rich diet and depends to a large extent on effectiveness of their alpha-amylases for survival. alpha-amylase (1-4-alpha-D-glucan glucanohydrolase) hydrolyses starch, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic linkages and has a major role in the utilization of polysaccharides. The recent increase in study of insect digestive enzymes seems to make sense in the realization that the gut is the major interface between the insect and its environment. Hence, an understanding of digestive enzyme function is essential when developing methods of insect control such as the use of enzyme inhibitor's and transgenic plants to control phytophagous insects. The aim of the current study is to identify and characterize alpha-amylase activity in order to gain a better understanding of its digestive physiology, which hopefully will lead to new strategies of the insect control. In order to analyze a-amylase activity adult and different nymphal stages were collected from wheat field from Karaj area and midgut complex from these individuals were dissected under a light microscope in ice-cold saline buffer (0.006M NaCl). After homogenization in buffer, homogenate was centrifuged at 15000 g for 20 min at 4 degrees C. The supernatant was pooled and stored at -20 degrees C for subsequent analysis. alpha-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure using soluble starch as substrate (starch 1%). Our result showed that enzyme activities in different nymphal stages (first, second, third, fourth and fifth stadium) were 0.19, 0.78, 1.21, 1.23, 1.25 units/mg protein, respectively.     

Age-specific digestion of Tenebrio molitor (Coleoptera: Tenebrionidae) and inhibition of proteolytic and amylolytic activity by plant proteinaceous seed extracts

Tenebrio molitor L. (Coleoptera: Tenebrionidae), is an international and serious pest of stored products. So far nothing is known about the activity for each growth stage digestive enzyme regarding this insect species. Thus, the aim of the current study was to get in depth analysis of the stage specific digestion and to investigate the effect of cereal (wheat cultivars including MV17, Aflak, Sivand, Saymon, and Zare) and legume (bean) seed extracts on the two main digestive enzymes i.e. α-amylases and proteases. Therefore, gut enzymes were extracted using distilled water and wheat cultivars and bean seed proteinaceous compounds were extracted using 0.1 M NaCl. Results showed that a steady state increase in the number and amount of digestive enzyme activities from first to fourth instar larvae was seen in both enzyme and in gel assays. In the first instar larvae (L1) only one band of α-amylase activity was seen (A1), whereas in the second (L2), third (L3), fourth (L4) and fifth (L5) instar larvae as well as in the adult (A) more than one amylase band (up to 4 isoenzymes) was seen. The same pattern was observed for α and β glucosidases and proteases. Probit analysis showed that bean and MV17 inhibited the amylase activity with an I₅₀ of 9.73 and 7.4 μg, respectively. The same cultivar seed extract inhibited protease activity with I₅₀s of 11.54 and 6.5 μg proteins. It is concluded that proteinaceous extract of cereals and bean seeds have a strong potential to be used in this pest management.     

Effect of PEG-induced drought stress on seed germination of four lentil genotypes

Seeds of four lentil genotypes (Castelluccio, Eston, Pantelleria, and Ustica) were subjected to five levels (0, 10, 15, 18, and 21%) of polyethylene glycol (PEG-6000). Germination percentage, root length, tissue water content (WC), α- and β-amylases, α-glucosidase activities, and osmolyte content were evaluated at 24, 48, and 72 h after starting the germination test. Water stress reduced seed germination percentage, root length, and seedling WC in all cultivars to different extent. The increase in proline content and total soluble sugars was greater for Eston and Castelluccio compared to the other genotypes. The activity of the enzymes involved in the germination process decreased in all cultivars; the activities of α-amylase and α-glucosidase were most negatively affected by osmotic stress, mainly in the drought sensitive Ustica and Pantelleria. Overall, Eston and Castelluccio were able to express greater drought tolerance and consequently could be used as a valuable resource for breeding programs.     

Alpha-amylase inhibitors from mycelium of an oyster mushroom

Abstract

The α-Amylase and α-glucosidase are two main enzymes involved in carbohydrate metabolism. This study was aimed at detecting alpha-amylase inhibitory activity from edible mushroom mycelia. Oyster mushroom was collected from a natural source, from Indian Institute of Technology (Banaras Hindu University) campus and was maintained in vitro in mycelial form. Chloroform, acetone, methanol, and water were used separately for extraction of an active constituent from mycelial cells grown, for 7?days, in potato dextrose broth. The extracts were tested for alpha-amylase inhibitory activity. Chloroform, acetone, and methanol extracts were found to have alpha-amylase inhibitory activity, with IC₅₀ values of 1.71, 224, and 383?μg/mL, respectively. Aqueous extract had no enzyme inhibitory activity. The acetone extract inhibited α-amylase non-competitively whereas chloroform extract showed competitive inhibition. Acetone extraction yielded highest total phenolic content (TPC) of 0.524?mM of gallic acid equivalent, whereas chloroform extraction resulted in lowest TPC of 0.006?mM. The HPLC and absorbance maxima of acetone and chloroform extracts suggest that the bioactive component responsible for enzyme inhibition could be glycoproteins in chloroform extract and catechins (flavonoids) in acetone extract. Thus, the mushroom mycelia under study may be exploited for production and purification of a lead compound for the development of the α-amylase inhibitory drug.     

New quinoxalinone inhibitors targeting secreted phospholipase A2 and α-glucosidase

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N′-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a–f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a–f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b–d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC₅₀?=?9.99?±?0.18 µM); which is comparable to quercetin (IC₅₀?=?9.93?±?0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC₅₀?>?200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.     

Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

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.     

The anthocyanins in black currants regulate postprandial hyperglycaemia primarily by inhibiting α-glucosidase while other phenolics modulate salivary α-amylase,glucose uptake and sugar transporters

The hypoglycaemic effects of two Ribes sp. i.e., anthocyanin-rich black currants (BC) were compared to green currants (GC), which are low in anthocyanins to establish which compounds are involved in the regulation of postprandial glycaemia. We determined the effect of the currants on inhibiting carbohydrate digestive enzymes (α-amylase, α-glucosidase), intestinal sugar absorption and transport across CaCo-2 cells. The digestion of these currants was modelled using in vitro gastrointestinal digestion (IVGD) to identify the metabolites present in the digested extracts by LC–MS/MS. Freeze-dried BC and IVDG extracts inhibited yeast α-glucosidase activity (P<.0001) at lower concentrations than acarbose, whereas GC and IVDG GC at the same concentrations showed no inhibition. BC and GC both showed significant inhibitory effects on salivary α-amylase (P<.0001), glucose uptake (P<.0001) and the mRNA expression of sugar transporters (P<.0001). Taken together this suggests that the anthocyanins which are high in BC have their greatest effect on postprandial hyperglycaemia by inhibiting α-glucosidase activity. Phytochemical analysis identified the phenolics in the currants and confirmed that freeze-dried BC contained higher concentrations of anthocyanins compared to GC (39.80 vs. 9.85 g/kg dry weight). Specific phenolics were also shown to inhibit salivary α-amylase, α-glucosidase, and glucose uptake. However, specific anthocyanins identified in BC which were low in GC were shown to inhibit α-glucosidase. In conclusion the anthocyanins in BC appear to regulate postprandial hyperglycaemia primarily but not solely by inhibiting α-glucosidase while other phenolics modulate salivary α-amylase, glucose uptake and sugar transporters which together could lower the associated risk of developing type-2 diabetes.     

Biochemical characterisation of digestive α-amylase of Red Palm Weevil,Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

The Red Palm Weevil, Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae), is a serious pest of a wide range of plant species including coconut, sago, date and oil palms. The α-amylases are the hydrolytic enzymes that are involved in carbohydrate metabolism in insects. So far nothing is done to demonstrate α-amylase activity of R. ferrugineus. Thus, the aim of the current study was to identify and characterise the α-amylase activity to gain a better understanding of digestive physiology of the insect. Thus, the α-amylase in the gut of red palm weevil was isolated and characterised using starch as a substrate. The study showed that the α-amylase is present in the gut of the insect for carbohydrate digestion. The α-amylase has an optimum pH and temperature of 5 and 40°C. The activity of α-amylase was increased by NaCl and KCl and inhibited by other compounds such as MgCl₂, CaCl₂, urea, ethylenediaminetetraacetic acid and sodium dodecylsulfate. Native-PAGE electrophoresis of α-amylase showed two isoenzymes, one major and one minor band showing α-amylase importance in the carbohydrate metabolism of the insect. Understanding of the digestive physiology and α-amylase activity of Red Palm Weevil is important when new management strategies for this economically important pest are devised.     

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, β-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas β-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.     

Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage.     

Effects of essential oil from Teucrium polium on some digestive enzyme activities of Musca domestica

The essential oil from Teucrium polium was evaluated for its adverse effects on larval instars of Musca domestica . The essential oil was blended with a diet at a concentration that produced 50% mortality of subjected insects (LC₅₀) (80 ppm). To learn about the situation of digestive enzymes of M. domestica treated with the essential oil, third larval instars were dissected under non-active enzyme conditions and their midguts were removed. The supernatant was used as an enzyme source after homogenization and centrifugation of the homogenates. Results revealed that some main digestive enzymes in the larval midgut were adversely affected when exposed to the food-incorporated essential oil. Proteinase extracted from larval midgut hydrolyzed the synthetic substrates B-Arg-pNA, Z-Arg-Arg-PNA and Z-Phe-Arg-PNA for trypsin, cathepsin B and cathepsin L activities, respectively, in control and treated larvae. The essential oil caused a reduction of 61.5% in tryptic activity. Significant 69% and 79% reductions were also observed in cathepsin L and B activities, respectively. Carbohydrase activities of α-amylase, α-glucosidase and β-glucosidase were detected in larval midgut extract. All assayed carbohydrases were affected by the essential oil. The most notable impact, a 93% reduction, was observed in α-amylase. Decreases of 69.5% and 42% were obtained in α-glucosidase and β-glucosidase activity, respectively. This study indicated that the larvicidal effect of the essential oil from Teucrium polium may be due to its detrimental effects on digestive enzymes. It seems that the detrimental effect of the oil can be due to both the inhibitory nature of the oil and the destruction of the midgut epithelium.