Effect of Ephestia kuehniella (Lepidoptera: Pyralidae) Larval Diet on Egg Quality and Parasitism by Trichogramma brassicae (Hymenoptera: Trichogrammatidae).

Trichogramma spp., among the most common parasitoids used for augmentation biological control, often are mass-reared on eggs of the Mediterranean flour moth, Ephestia kuehniella (Zeller). To evaluate removal of nutritional components from the E. kuehniella larval diet and reduce production costs, colonies were maintained using one of three diets: a standard diet consisting of eight ingredients, a reduced diet containing whole wheat flour, glycerol, and Brewer's yeast, or a third minimal diet of only whole wheat flour. The standard diet sustained the fastest larval development, female pupae with the greatest mass, the highest level of adult emergence, and production of the most eggs per female. Eggs from moths reared as larvae on the standard or reduced diet had equivalent mass, length, and percent hatch. Females from larvae fed the minimal diet produced eggs with the least mass that were shorter and had the lowest percent hatch. Eggs from the three E. kuehniella colonies were exposed separately to Trichogramma brassicae Bezdenko females to determine their acceptance for oviposition. More of the eggs from the standard diet were parasitized by the females, eggs from the reduced and minimal diets being less acceptable. The percent emergence of the parasitoids was the same regardless of diet; however, the largest wasps emerged from the standard diet eggs and a greater proportion of them were females. Consequently, the standard E. kuehniella larval diet resulted in the highest rate of reproduction and robust eggs that produced superior T. brassicae wasps.

MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting.

Role of symbiotic bacteria in the growth and development of the Sunn pest, Eurygaster integriceps.

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is the most important pest of wheat and barley in wide areas of the world. Different aspects of the insect's life history have been studied, but to date nothing is known about their microbial symbionts. Here, the contribution of symbiotic bacteria to the fitness of the bug was investigated by combining two different approaches to manipulate the host's microbial community: the supplementation of antibiotics into the insects' diet and egg surface sterilization. First, bacteria cultured from gut homogenates were subjected to antibiotic screening tests using 20 different antibiotics. Norfloxacin was the most effective antibiotic, with the greatest inhibition zone among all antibiotics tested. Feeding norfloxacin to adult E. integriceps individuals significantly impaired growth and development of the offspring in a dose-dependent manner, i.e., higher antibiotic doses increased the negative effects on nymphal growth and development. Total developmental time from first nymphal instars to adult emergence in control animals was 30.1 days, but when adults had been offered diets with 10, 20, and 30 µg antibiotic per mg diet, the offspring's developmental time was prolonged to 32.8, 34.0, and 34.8 days, respectively. In the highest two doses of norfloxacin, all of the nymphs died before reaching the fifth nymphal instar. Similar results as for the antibiotic treatment were obtained when egg surface sterilization was used to manipulate the microbial community of E. integriceps. These results indicate that bacterial symbionts play a crucial role in the successful development of the host.

Secretion and formation of perimicrovillar membrane in the digestive system of the Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in response to feeding.

In this study, development of perimicrovillar membrane (PMM) from midgut cells of starved and fed Eurygaster integriceps (Hemiptera: Scutelleridae) was studied. Three different approaches, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), marker enzymes of the PMMs (α-glucosidase), perimicrovillar space (aminopeptidase), and microvillar membranes (ß-glucosidase) were used. Activities of these enzymes were remarkably low in the starved insects. Moreover, microscopic observations revealed that PMM is not present in the starved insect. Activities of enzymatic markers increased at 5 h postfeeding, and TEM and SEM observations showed the formation of PMM as well as migration of double-membrane vesicles from center of the columnar cell to the cell apex. The highest PMM was observed at 20 h postfeeding which at this time marker enzyme activity, such as α-glucosidase activity, was high, too. Thus, at 20 h postfeeding, PMM system was evident and epithelial cells were completely covered by PMM system. After 20 h postfeeding, presence of the fine holes in PMM started to be seen and at 40 h post-feeding, observation showed degradation of PMM system. Thus, it could be concluded that PMM in E. integriceps is secreted by epithelial cell membrane when needed and its secretion and formation is regulated by feeding. This system was not present in the starved insects as its development takes place at 5 h postfeeding.

A trypsin-like protease in rice green semi-looper, Naranga aenescens Moore (Lepidoptera: Noctuidae): purification and characterization.

The rice green semi looper, Naranga aenescens Moore (Lepidoptera: Noctuidae) causes severe damage to rice fields in Eastern Asia and Middle East. We demonstrate that two types of serine proteases are active in the midgut of the third instar larvae of N. aenescens, but trypsin-like proteases are considerably more active than chymotrypsin-like proteases. To develop better control strategies, purification and biochemical characterization of a major trypsin-like digestive protease from the midgut of the third instar larvae of N. aenescens was achieved by gel filtration and anion exchange chromatography. After the final purification step, the enzyme was purified 9.62-fold with a recovery of 16.1% and a specific activity of 4.12 U/mg protein and a molecular mass of approximately 88.5 kDa. Biochemical characterization indicated that the purified protease had highest activity at pH 10 and 30°C and was stable for up to 6 h under those conditions. Divalent cations, especially Ca2+, Mg2+, and Cu2+, increased the enzyme activity and synthetic inhibitors that target trypsin-like activity caused a significant reduction in caseinolytic activity. These data may be used to develop inhibitors that decrease the damage of N. aenescens to rice cultivars in the field.

Cellular immune reactions of the sunn pest, Eurygaster integriceps, to the entomopathogenic fungus, Beauveria bassiana and its secondary metabolites.

In this study, five morphological types of circulating hemocytes were recognized in the hemolymph of the adult sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), namely prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids. The effects of the secondary metabolites of the entomopathogenic fungus Beauveria bassiana on cellular immune defenses of Eurygaster integriceps were investigated. The results showed that the fungal secondary metabolites inhibited phagocytic activity of E. integriceps hemocytes and hampered nodule formation. A reduction of phenoloxidase activity was also observed. The data suggest that B. bassiana produce secondary metabolites that disable several immune mechanisms allowing the fungus to overcome and then kill its host. This characteristic makes B. bassiana a promising model for biological control of insect pests such as E. integriceps.

A study of the changes in quantitative activity of some enzymes in olive cultivars during the interaction between Verticillium wilt and root-knot nematode.

Second stage juvenile (J2) of root-knot nematode, Meloidogyne javanica, and microsclerotia of verticilliosis, Verticillium dahliae, were used as the source of inoculum for nematode and fungus respectively. One-year-old seedlings of olive cultivar, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. Experiment was conducted in completely randomized design with 32 Treatments and five replications. Treatments were as follows: control, nematode alone, fungus alone and fungus+nematode. Pots were inoculated with (0, 2000, 3000, 4000) J2 of nematode and/or (10 no/g soil) microsclerotia of fungus according to the treatments. Quantitative activity of soluble proxidase, cell wall bounded proxidase, beta-1,3-glucanase and B-1,4-glucanase were determined by the pirocatechol, hydrogen proxide, guaiacol and laminarin-dinitrosalicylilate methods, respectively, on 1,10, 20 and 30 days after inoculation. Results showed that these enzymes in leaves and roots of seedlings inoculated with pathogens increased (p < or = 0/05). In this study changes of these enzymes as compared with nematode alone and fungus alone treatments were increased in fungus+nematode treatments in seedlings (p < or = 0/05). Maximum of quantitative changes of these enzymes were observed in fungus+nematode (4000J2) treatment on cv. Koroneiki. Based on the results obtained in this study, quantitative activity of these enzymes in olive roots and leaves were be more on cvs. Koroneiki, Roghani, Zard and Manzanilla, respectively (p < or = 0/05).

A study of interaction between Verticillium wilt Verticillium dahliae and root-knot nematode Meloidogyne javanica in olive cultivars.

Second stage juvenile (J2) of root-knot nematode, Meloidogyne javanica, and microsclerotia of verticillium wilt, Verticillium dahliae, were used as the source of inoculum for nematode and fungus respectively. One-year-old seedlings of olive cultivars, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. Experiment was conducted in completely randomized design with 32 treatments and five replications. Treatments were as follows: control, nematode alone, fungus alone and fungus + nematode. Pots were inoculated with (0, 2000, 3000, 4000) J2 of nematode and/or (10 no/g soil) microsclerotia of fungus according to the treatments. Experiment was terminated after 10 months and fallowing parameters were determined i.e., fresh weight of root and stem, number of galls and egg masses per root system, and percentage of incidence of symptom on aerial parts, browning of vascular tissue, decrease of seedling height and stem/root tissue colonization by fungus. Results showed that presence of nematode caused reduction on colonization of the fungus in the root and stem and vice versa i.e. presence of fungus caused reduction on number of galls and egg masses produced by the nematode. Severe fungus wilt on aerial parts of Manzanilla cultivar was observed when both pathogens were inoculated and mild fungus wilt was observed in fungus alone treatments of Koroneiki cultivar. Galling and egg mass production in root system were reduced in cvs Manzanilla, Zard, Roghani and Koroneiki, respectively (p < or = 0/05). Based on the results obtained in this study, verticilliosis symptoms and galling of nematode in olive seedlings were be less on cvs Koroneiki, Roghani, Zard and Manzanilla, respectively (p < or = 0/05).

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae).

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/ß-mannosidases) were little in comparison to activities against glucose (α/ß-glucosidases) and galactose (α/ß-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.

Purification and characterization of phenoloxidase from the hemocytes of Eurygaster integriceps (Hemiptera: Scutelleridae).

Insects protect themselves from microbial invaders by two main immune activities, namely the cellular and humoral reactions. Phenoloxidases are oxidative enzymes that have an important role in both cell-mediated and humoral immunity. In this study, the purification and biochemical characterization of a phenoloxidase from the hemocytes of the Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) were carried out. After the final purification step, the enzyme was purified 7.31-fold with a recovery of 3.94% and a specific activity of 4.95U/mg protein. Results of the biochemical characterization showed that the purified phenoloxidase has a maximum activity at pH 6 and at 30-35°C and is stable for 24-36h. Divalent cations such as Ca(2+) and Cu(2+) significantly increased the enzymatic activity and synthetic inhibitors such as phenylthiourea significantly decreased it. The purified phenoloxidase has a molecular mass of 22kDa. The current paper represents a further step towards the characterization of humoral immunity of E. integriceps in order to develop new strategies for the biological control of the Sunn pest.

Enzymatic properties of alpha- and beta-glocusidases extracted from midgut and salivary glands of rice striped stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae).

The study of digestive enzymes, especially in important pests like Chilo suppressalis Walker (Lepidoptera: Pyralidae), which are a key constraint on rice production in a wide area of the globe and also in Iran, could be a successful procedure in the development of a safe and useful control strategy. Glycosidase are a type of digestive enzymes which have a critical role in the final stages of carbohydrate digestion; they hydrolyze alpha-D-(1,4)-glucose linkage such as p-nitrophenyl-alpha-D-glucoside in di and oligosaccharide components. Laboratory reared 4th instar larvae were randomly selected; midgut and salivary gland were removed by dissection under a stereo microscope and glucosidase activities were assayed by Ferreira and Terra's procedures. The activities of alpha- and beta-glucosidase in the midgut and salivary gland were 0.009, 0.0063, 0.005 and 0.003 micromol/min/mg protein, respectively. The optimal pH and temperature for enzyme activity were determined to be 9 and 45 degrees C for the glucosidases measured, values which are in agreement with other reports, especially in lepidopteran insects, which give values between 8-12 and 20-50 degrees C. The enzyme activity increased with the addition of NaCl, MgCl(2) and CaCl(2) and decreased due to the use of different concentrations of KCl, Urea, EDTA, SDS and Urea both in midgut and the salivary glands. Control of pests by using resistant varieties is one of the most important practices that are dependent on inhibitors in plants. Hence, characterization of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful, on the one hand for a better understanding of enzyme roles in the nutrition physiology of insects, and on the other hand to reach safe and useful controls of insect pests.