Temperature and Ca2+ ion as modulators in cellular immunity of the Sunn pest Eurygaster integriceps Puton (Heteroptera: Scutelleridae)

Arash ZIBAEE,Ali Reza BANDANI,Reza TALAEI-HASSANLOUEI,Davide MALAGOLI 한국곤충학회 2009 Entomological Research Vol.39 No.6

Environmental conditions in addition to divalent cations may affect the interactions between pathogens and insects. Elucidation of factors which modulate insect immune responses could be a significant part of investigations in this area. In this study, adults of Eurygaster integriceps, as the destructive pest of wheat, were kept at different temperatures in addition to injection with different concentrations of Ca2+ to find the effect on cellular immune reactions against Beauveria bassiana. Results showed that total and differentiate hemocyte numbers, nodule formation and phenoloxidase activity increased with elevation of temperature so that the higher values were obtained at 30 and 40°C at various intervals. Higher concentrations of Ca2+ ion (5 mM) caused an increase in plasmatocyte length and width especially after 60 min. Similar results were observed for nodule formation and phenoloxidase activity of E. integriceps adults after injection by B. bassiana spores and phenoloxidase activity. It is clear from the current study that thermoregulation and Ca2+ ion can positively affect the hemocyte numbers especially plasmatocytes and granulocytes, nodule formation and phenoloxidase activity in E. integriceps. The understanding of modulators of the insect immune response may directly influence novel approaches to obtain safe and effective biological control agents.

Proteolytic activity in Plagiodera versicolora Laicharting (Coleoptera: Chrysomelidae): Characterization of digestive proteases and effect of host plants

Arash Zibaee,Jalil Hajizadeh 한국응용곤충학회 2013 Journal of Asia-Pacific Entomology Vol.16 No.3

Proteolytic profiles in the midgut of Plagiodera versicolora were studied using biochemical approaches, and the effects of host plants on possible changes in their activity were determined. Morphology of the alimentary canal revealed several areas of sections, namely bucca, pharynx, esophagus, crop, midgut, ileum, rectum and anus. A pH of 6 and 11 was found to be optimal for soluble and membrane-bound fractions, by using azocasein 2% as a substrate. Determination of specific proteases demonstrates the presence of trypsin-like,chymotrypsin-like, elastase, cathepsin B, cathepsin L and cathepsin D, as well as two exopeptidases. Regarding site of activity for each specific protease, it was found that the major activity of cathepsin B and cathepsin L was in the soluble fraction, chymotrypsin, cathepsin D and two exopeptidases in membrane-bound fraction. Additionally, trypsin-like and elastase activities had no significant differences between fractions. The presence of the above mentioned specific proteases was verified using the specific inhibitors PMSF, TLCK, TPCK, cystatin,phenanthroline and DTT. Feeding of the beetle on four host plants: including Salix aegyptica, S. alba, Populus alba and P. caspica, from the 1st larval instar to adult, revealed the highest trypsin-, chymotrypsin-like and elastase activities in the individuals fed on S. aegyptica and S. alba, respectively. Regarding cathepsins B and L, the highest activities were observed on S. alba and S. aegypticum but cathepsin D was higher in S. Alba and P. alba. Feeding on S. alba and S. aegypticum showed the highest activities of amino- and carboxy-peptidases, respectively.

Characterization of α-amylase in the midgut and the salivary glands of rice striped stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Arash Zibaee,Ali Reza Bandani,Maryam Kafil,Samar Ramzi 한국응용곤충학회 2008 Journal of Asia-Pacific Entomology Vol.11 No.4

The rice striped stem borer, Chilo supprressalis, is a destructive pest of rice that was introduced to Iran in 1973 and has since become widely distributed. Amylases are hydrolytic enzymes that catalyze the hydrolysis of the α-D-(1,4)-glucan linkage in glycogen and other related carbohydrates. Laboratory-reared 4th stadium larvae were randomly selected; the midgut and the salivary glands were removed by dissection under a dissecting microscope and α-amylase activity was assayed using the dinitrosalicylic acid procedure. The activity of α-amylase in the midgut and salivary gland were 0.06 and 0.036 μmol/min/mg protein, respectively. The optimal pH and temperature for α-amylase were 9 and 35–40 °C, which is consistent with reports of other lepidopteran insects. The enzyme activity was inhibited by addition of NaCl, KCl, MgCl2, Urea, EDTA, and SDS. CaCl2 enhanced enzyme activity. Ca2+ ions also had inhibitory effects on glucosidase activity. Plant amylase inhibitors play important role against insect pests. Hence, the characterization of digestive enzymes and the examination of inhibitors on enzyme activity could be useful in tackling of insect pests. The rice striped stem borer, Chilo supprressalis, is a destructive pest of rice that was introduced to Iran in 1973 and has since become widely distributed. Amylases are hydrolytic enzymes that catalyze the hydrolysis of the α-D-(1,4)-glucan linkage in glycogen and other related carbohydrates. Laboratory-reared 4th stadium larvae were randomly selected; the midgut and the salivary glands were removed by dissection under a dissecting microscope and α-amylase activity was assayed using the dinitrosalicylic acid procedure. The activity of α-amylase in the midgut and salivary gland were 0.06 and 0.036 μmol/min/mg protein, respectively. The optimal pH and temperature for α-amylase were 9 and 35–40 °C, which is consistent with reports of other lepidopteran insects. The enzyme activity was inhibited by addition of NaCl, KCl, MgCl2, Urea, EDTA, and SDS. CaCl2 enhanced enzyme activity. Ca2+ ions also had inhibitory effects on glucosidase activity. Plant amylase inhibitors play important role against insect pests. Hence, the characterization of digestive enzymes and the examination of inhibitors on enzyme activity could be useful in tackling of insect pests.

Proteolytic profile in the larval midgut of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Arash Zibaee 한국곤충학회 2012 Entomological Research Vol.42 No.3

Chilo suppressalis is a key constraint on production of rice. The current research was conducted to study the types of digestive proteases in the larval midgut of C. suppressalis. It was found that activity of total digestive proteases increased from the first to the fifth larval instars, which showed different nutritional requirements. Four types of proteinases and two types of exopeptidase were identified so that their activities from the highest to the lowest activities is trypsin‐like, chymotrypsin‐like and elastase for proteinases, and amino and carboxypeptidases for exopeptidases. Meanwhile, just one type of cysteine protease, cathepsin D, was determined in the fourth and fifth instar larvae. The optimal pH for activity of total protease was found to be pH 9–10 and optimal temperature was observed to be 35–40°C, where there was the highest proteolytic activity. Some specific inhibitors of proteases including PMSF, TLCK, TPCK, DTT, E‐64, cystatin, phenanthroline and EDTA were used to confirm the types of proteases in the midgut of C. suppressalis.

Effects of Citrullus colocynthis agglutinin on intermediary metabolism of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae)

Samar Ramzi,Ahad Sahragard,Arash Zibaee 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.3

In the current study,we examined the effects of Citrullus colocynthis agglutinin (CCA) on the intermediary metabolismofEctomyelois ceratoniae larvae as a means of determining effectiveness of the lectin in pest control. Larvaewere fed on control diets and diets containing CCA (2%) for 24 and 48 h. The activities of digestive α-amylase andgeneral proteases significantly decreased in the larvae fed on the CCA diet, but no statistically significant changeswere found in the case of TAG-lipase. Meanwhile, the activities of all specific proteases, except aminopeptidase,changed in the treated larvae for all time intervals. The activity of alanine aminotransferase significantly decreasedin the treated larvae but the activities of aspartate aminotransferase and γ-glutamyl transferase increased24 h post-feeding on the CCA diet. The activity of aldolase decreased 48 h post-feeding but lactatedehydrogenase (LDH) showed inverse results at the same time interval. The activities of acid phosphatase(ACP) and alkaline phosphatase (ALP) decreased 48 h post-feeding for ACP and at both time intervals for ALP. The amount of high-density lipoprotein (HDL) showed no statistical differences between control and CCA-fedlarvae, but the amount of low-density lipoprotein (LDL) significantly increased in the CCA-fed larvae at bothtime intervals. The amount of protein increased in the larvae fed on the CCA diet, but the amounts of glycogenand triacylglycerol significantly decreased at both time intervals. Results of the current study clearly revealthat ingestion of CCA significantly influences the activities of digestive enzymes and disrupts the intermediarymetabolism of E. ceratoniae larvae. Further studies on genetic engineering of CCA to provide resistant varietiesmight open a new avenue for safe and efficient control of the pest.

Digestive proteolytic activity in larvae and adults of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Sahar Delkash-Roudsari,Arash Zibaee,Mohammad Reza Abbci-Mozhdehi 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.3

Digestive proteolytic activity in larvae and adults of Bactrocera oleae was studied using specific substrates andinhibitors. The optimal pH for general proteolytic activity was 4 and 10 for soluble and membrane-bound fractionsof larvae, and 9 for the soluble fraction of adults. The highest activities of general proteases were revealed attemperatures of 25 °C and 45 °C for both the soluble andmembrane-bound fractions of larvae as well as the solublefraction of adults. Determination of the specific protease activities demonstrated the presence of serine and cysteineproteases in addition to two exopeptidases in the larvae and adults. However, trypsin-like protease, chymotrypsinlikeprotease, and two exopeptidases of larvae, and chymotrypsin-like protease as well as cathepsin L of adults hadno activity in the soluble fraction. The presence of specific proteases was verified by using specific inhibitors such asPMSF, TLCK, TPCK, E-64, EDTA, phenanthroline, and DTT. Finally, feeding of B. oleae larvae on different olive varietiesrevealed the highest trypsin-like protease, chymotrypsin-like protease, elastase, cathepsin B, cathepsin L, andcathepsin D on Amigdalifolia, Coratina, Baladi, Mari, Conservalia, Baladi, and Arbequina, respectively. These resultsshowed digestive proteolytic activities in B. oleae for the first time, and could be the basic knowledge required forfinding a control procedure to decrease the damage of this destructive pest around the world.

Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae)

Yousefi-Lardeh Leila,Zibaee Arash 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.4

Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V max of PO was obtained using L-dopa while the lowest K m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC 50 of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K m . Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.

Comparison of glutathione S-transferase and general esterase in two bugs, Eurygaster integriceps Puton (Heteroptera: Scutelleridae) and Brachynema germari Kolenati (Heteroptera: Pentatomidae)

Samar RAMZI,Ali Reza BANDANI1,Arash ZIBAEE,권오석 한국곤충학회 2009 Entomological Research Vol.39 No.4

Glutathione S-transferases (GSTs) and general esterases play important roles in the detoxification of many substances including allelochemicals from plants and chemical pesticides. In this study, to determine the feeding status and effects of agronomical practices on GST and general esterase, two bug species, Eurygaster integriceps and Brachynema germari, were selected with differences in these cases. GST and general esterase responses varied when switching from 1-chloro-2,4-dinitrobenzene (CDNB) and a-naphtyl to 1,2-dichloro-4-nitro-benzene (DCNB) and b-naphtyl in E. integriceps and B. germari, respectively. The activities of GST and esterase by using CDNB and a-naphtyl were higher than that of DCNB and b-naphtyl for both insects. It was shown that the optimal pH for GST and general esterase activity varied between pH 5.5 and 6. While the Michaelis constant (Km) value related to E. integriceps toward CDNB and a-naphtyl was lower than Km for B. germari, Km values calculated for E. integriceps toward DCNB and b-naphtyl corresponding to those related to A. hejeri were similar to CDNB and a-naphtyl values. The maximal reaction velocity (Vmax) values related to E. integriceps for both substrates (CDNB and a-naphtyl) were always higher than those from B. germari and showed significant differences. At the basement of the native-PAGE electrophoresis, five bands were observed in for GST and three were visualized for B. germari, with a large, darker band for E. integriceps in the case of esterases. Studies on the detoxification enzymes of herbivores should be undertaken to determine accurately the effect of the host plants on the organisms eating them, particularly in terms of biochemical and ecological advantages.

Effect of pyridalyl on mortality, fecundity and physiological performance of olive fruit fly, Bactrocera oleae Rossi (Diptera: Tephritidae)

Mohammad Reza Abbasi-Mojdehi,Jalil Hajizadeh,Arash Zibaee,Ali Akbar Keyhanian 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2

Toxicity and physiological effects of pyridalyl were studied on the adults of Bacterocera oleae via oral exposure and biochemical experiments. First, adults were fed on a protein hydrolysate diet which has been amended by five concentrations of pyridalyl. Then, another cohort of flies was fed on LC 50 concentration to find potential effects on fecundity, detoxifying enzymes and intermediary metabolism. Results of bioassay showed a concentration of 0.517 μg/ml as the LC 50 value with confidence limit of 0.305–0.791 μg/ml at 95%. Fecundity of the females fed on pyridalyl-treated diet showed no significant differences with controls following 7 days of exposure while a significant higher mortality was recorded in laid eggs by the treated females. Activities of esterase and glutathione S-transferase significantly increased in the treated adults compared to control when α-naphthyl acetate and CDNB (1-chloro-2,4-dinitrobenzene) were used as substrates. Activities of alanine- and aspartate aminotransferases significantly increased in the pyridalyl-treated adults compared to control while no significant difference was observed in activity of ɤ-glutamyl transferase. Although lactate dehydrogenase showed no statistically different activity but both acid- and alkaline phosphatases had statistically higher activity in the control adults compared to treatment. Finally, low density lipoprotein and total protein were the only non-enzymatic components which showed statistical difference between control and treated flies. Our results indicated that pyridalyl had toxicity on B. oleae and intervened in the physiological performance of flies.