Pectinase and cellulase activity in the digestive system of the elm leaf beetle, Xanthogaleruca luteola Muller (Coleoptera: Chrysomelidae)

Mohammad VATANPARAST,Vahid Hosseininaveh,Mohammad Ghadamyari,Seyede Minoo Sajjadian 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.4

The elm leaf beetle, Xanthogaleruca luteola, is a serious pest of elm trees in urban areas. Partial biochemical characterization of pectinases and cellulases was conducted using the larval digestive system of the pest. Midgut extracts from larvae showed optimum activity for pectinase and cellulase against pectin and carboxymethyl cellulose, respectively, under acidic conditions (pH 6). Pectinases and cellulases were respectively more stable under acidic conditions (pH 4–7) and slightly acidic conditions (pH 5–7) than under highly acidic and alkaline conditions. However, the enzymes were more stable in slightly acidic conditions (pH 6) when incubation time was increased. Maximum activity for the pectinases and cellulases incubated at different temperatures was observed at 45 and 50 °C, respectively. Mg2+ remarkably increased pectinase activity,and cellulase activity increased significantly in the presence of Ca2+ and Mg2+. Sodium dodecyl sulfate significantly decreased pectinase and cellulase activity. The Michaelis–Menten constant (KM) and the maximal reaction velocity (Vmax) values for pectinase were 2 mg·mL−1 and 0.017 mmol·min−1·mg−1 protein toward pectin, respectively. Zymogram analyses revealed the presence of one and five bands of pectinase and cellulase activity, respectively, in the larval midgut extract.

Digestive proteolytic activity in the pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Mahdieh Bigham,Vahid Hosseininaveh 한국응용곤충학회 2010 Journal of Asia-Pacific Entomology Vol.13 No.3

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators,respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m1 and m3,respectively, and maximum chymotrypsin and trypsin activities were observed in m3. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.

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

Samar Ramzi,Vahid Hosseininaveh 한국응용곤충학회 2010 Journal of Asia-Pacific Entomology Vol.13 No.3

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.

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

Mahdieh BIGHAM,Vahid HOSSEININAVEH,Banafsheh NABAVI,Khalil TALEBI,Naimeh-Sadat ESMAEILZADEH 한국곤충학회 2010 Entomological Research Vol.40 No.1

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.

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

Mohammad VATANPARAST,Vahid HOSSEININAVEH 한국곤충학회 2010 Entomological Research Vol.40 No.6

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive -amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for -amylase against starch at acidic pH (pH 5.0). -Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. -Amylase activity was significantly decreased in the presence of Ca2+, Mg2+ and sodium dodecylsulfate. On the contrary, K+ and Na+ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of -amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for -amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.

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

Parvin Razavi Tabatabaei,,Vahid Hosseininaveh,Seyed Hossein Goldansaz,Khalil Talebi 한국응용곤충학회 2011 Journal of Asia-Pacific Entomology Vol.14 No.2

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. Proteasespecific 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.