Dietary Protein and Carbohydrate Levels Affect Performance and Digestive Physiology of Plodia interpunctella (Lepidoptera: Pyralidae).

In this study, life history and nutritional indices of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) was evaluated on six food commodities: dried fig, dried wheat germ, dried white mulberry, groundnut, pistachio, and raisin, compared with artificial diet. The influence of dietary macronutrient content on digestive α-amylase was also assessed. A delay in the developmental time of P. interpunctella immature stages was detected when larvae were fed on raisin. The highest survival rate of immature stages was on the artificial diet, and the lowest was on raisin. The highest realized fecundity and fertility were recorded for the females reared on artificial diet. Overall, fourth instar P. interpunctella reared on artificial diet had the highest relative consumed and growth rate, and lowest rates were observed in larvae fed raisin. Amylolytic activity and isoform patterns varied depending on larval instar and diets, but were higher for larvae fed artificial diet with moderate carbohydrate and protein. Zymograms showed the presence of three isoforms of α-amylase in midgut extracts of P. interpunctella fed different diets. Larvae fed dried white mulberry, fig, and raisin had one (A2) α-amylase isoform. The data suggest that dietary carbohydrate and protein content induce changes in nutritional efficiency, development, and α-amylase activity. A survey of the differences in digestive enzyme activity in response to macronutrient balance and imbalance highlight their importance in the nutrition of insects.

Inhibitory effects of an extract from non-host plants on physiological characteristics of two major cabbage pests.

The diamondback moth (Plutella xylostella) and small white cabbage butterfly (Pieris rapae) are the two main serious pests of cruciferous crops (Brassicaceae) that have developed resistance to chemical control methods. In order to avoid such resistance and also the adverse effects of chemical pesticides on the environment, alternative methods have usually been suggested, including the use of plant enzyme inhibitors. Here, the inhibitory effects of proteinaceous inhibitors extracted from wheat, canola, sesame, bean and triticale were evaluated against the digestive α-amylases, larval growth, development and nutritional indecs of the diamondback moth and small white cabbage butterfly. Our results indicated that triticale and wheat extracts inhibited α-amylolytic activity in an alkaline pH, which is in accordance with the moth and butterfly gut α-amylase optimum pH. Dose-dependent inhibition of two crucifer pests by triticale and wheat was observed using spectrophotometry and gel electrophoresis. Implementation of specificity studies showed that wheat and triticale-proteinaceous extract were inactive against Chinese and purple cabbage amylase. Triticale and wheat were resistant against insects' gut proteases. Results of the feeding bioassay indicated that triticale-proteinaceous extract could cause a significant reduction in survival and larval body mass. The results of the nutritional indecs also showed larvae of both species that fed on a Triticale proteinaceous inhibitor-treated diet had the lowest values for the efficiency of conversion of ingested food and relative growth rate. Our observations suggested that triticale shows promise for use in the management of crucifer pests.

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): enzyme markers of microvillar and perimicrovillar membranes.

The subcellular distributions of six digestive and non-digestive enzymes (alpha-glucosidase, beta-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and alpha-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and beta-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of alpha-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that beta-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from beta-glucosidase carrying membranes is different from that of alpha-glucosidase carrying membranes. We conclude that beta-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while alpha-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes.

Effects of Artemisia annua L. (Asteracea) on the digestive enzymatic profiles and the cellular immune reactions of the Sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauveria bassiana.

Plant extracts are currently studied more and more because of the possibility of their usage in plant protection. Many of the natural plant compounds which are used in the control of pests are known to affect the digestion and immune functions of insects. In this study, effects of Artemisia annua extract on the digestive enzymatic profiles and the cellular immune reactions of Eurygaster integriceps were investigated to reach a better understanding of its role in the control of this pest as the most destructive one in the production of wheat in the Near and Middle East, eastern and southern Europe and North Africa. Feeding and injection methods were used to study the plant extract effects on digestive enzymes and cellular immunity, respectively. When adult E. integriceps fed on food and water containing plant extracts, activity of the digestive enzymes, including alpha-amylase, alpha- and beta-glucosidases, protease and lipase, in addition to cellular immune reactions (total and differentiate hemocyte numbers, phagocytosis, nodule formation and phenoloxidase activity) against Beauveria bassiana were affected and significantly decreased in comparison with controls, in that the clear dose-response relationships were established with respect to enzyme activities and immune reactions. A. annua extract had a significant effect on kinetic parameters (Vmax and Km) of digestive enzymes and phenoloxidase activity so that the presence of the plant extract decreased the value of Vmax and increased Km, causing the reduction of enzyme affinity to the substrate, overall velocity of the reaction and finally interfering with the rate of breakdown of the enzyme-substrate complex. The understanding of fungal-induced immune responses and identification of factors regarding fungal virulence could be important in accelerating host death in a biological control scenario. Hence, the combination of botanical pesticides and microbes to control insect pest populations would be a safe and possibly rapid method to decrease their damage and environmental risk due to the use of chemical pesticides.

Effect of some diets on Macrolophus pygmaeus rambur (Hemiptera: Miridae) fitness under laboratory conditions.

Fitness parameters of omnivore predatory bug Macrolophus pygmaeus were studied on various diets consisting of green peach aphid Myzus persicae + eggplant leaf disc, eggs of Ephestia kuehniella + eggplant leafdisc, pollen + eggplant leaf disc, eggs of E. kuehniella + pollen + eggplant leaf disc. The experiments were began from 1- day nymphs until 32-day adults. The tests were done at 25 degress C, 65+/-5% RH, 16L: 8D h photoperiod. The results showed that adding floral material to the animal prey (eggs of E. kuehniella) containing treatment shortened the nymphal development time. The highest rate of mortality of M. pygmaeus nymphs was observed on pollen + eggplant leaf disc. The sex ratio of emerged adults was similar between treatments. The preoviposition period was the same in a ll treatments. T he fecundity of M . pygmaeuswas affected by feeding diets. Females fed on eggs of E. kuehniella + pollen + eggplant leaf disc have highest rate of fecundity. Based on results, diet of E. kuehniella eggs + pollen + eggplant leaf disc is the most suitable diet for rearing of this predatory insect. Obtained results somehow were expectable due to the high nitrogen extent, vitamins and mineral materials found in the pollen.

Oil formulation of entomopathogenic fungus, Beauveria bassian, against Sunn pest, Eurygaster integriceps puton (Heteroptera: Scutelleridae).

Entomopathogenic fungi with approximately 1000 known species are key regulatory factors in insect pest population. Also, these agents infect insects by direct penetration of the cuticle and thus provide the only practical means of microbial control of insects that feed on plant or animal juices as well as for the many coleopterans and orthopteran pest which have very few known viral or bacterial diseases. Beauveria bassiana is a widespread entomopathogenic fungi that infect many insect species. The aim of the current study was to determine the effect of different composition of oil in pathogenicity of the fungus. So, the fungus was grown on Potato dextrose agar (PDA) at 25 degrees C. Conidia were harvested from 14-day sporulating cultures by scraping the surface with a spatula and suspending the conidia in sterile 0.03% aqueous Tween 80. The number of conidia was determined using an improved Neubauer haemocytometer and spore concentrations of 10(4), 10(5), 10(6), 10(7), 108 and control were prepared. Two controls were done one was different concentrations of spore without oil and the other only oil. The insects were collected from the wheat farm in and maintained on wheat plants in the laboratory at 27+/-2 degrees C under a 14 h light: 10 h dark (LD 14:10) photoperiod. Assays were done with dipping methods. The results showed that there are significant differences in mortality of insects in two spore emulsions, one oil formulated and the other spores in sterile water without oil usage. Oil formulation enhances fungal virulence toward insect. Since insect cuticle especially epicuticle (lipid layer) is the primary site of establishment of mycosis, oil formulation increase the adhesion of spore to the insect cuticle through hydrophobic interaction between the spore and cuticle surface.

Comparison of alpha-amylase activity in larval stages of flour beetles, Tribolium confusum (Coleoptera: Tenebionidae).

Flour beetles attack stored grain products such as flour, cereals, meal, dried pet food, dried flowers and even dried museum specimens and other foods in the house. Stored-product insects cause tremendous losses by lowering weight, germination rate, nutritional value and grain grade. These beetles are of the most important pests of stored products in the home and grocery stores. The adult female may live for as long as two years, depositing 300 to 400 eggs. The life cycle requires one to four months when temperatures are favorable. Several methods could be used to control this insect including synthetic insecticides, biological control, physical control and transgenic plant carrying gene of interest. Chemical controls are discouraged due to pesticide residue in the commodities and resistance in insects. The 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 inhibitors and transgenic plants to control insect pests. Therefore, the aim of the current study was to get a good understanding from enzyme composition of different larval stages of the insect and finally characterize amylase which is the key enzyme in digestive system of this insect. For alpha-amylase study whole larvae were homogenized in 0.02 M phosphate buffer at pH 7.2. The homogenates were separately transferred to a 1.5 ml of centrifuge tubes and centrifuged at 15000xg for 20 min at 4degrees C. The supernatants were used as enzyme source in assays. alpha-Amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure using 1% soluble starch (Merck) as substrate. The results show that enzyme activity (OD) in the first, second, third and fourth larval stages were 0.5, 1.15, 1.35 and 1.362, respectively. There are significant differences in amylase activity in different larval stages; however, there are no significant differences in the enzyme activity of two last larval stages. Wheat grain is a major source of starch and insect that feed on the wheat grain or flour made from wheat rely heavily on their amylase for starch hydrolyze and this could be the main reason that larval stages even first larval instar has such a high amount of alpha-amylase.

Comparison of pathogenicity of two isolates of the entomopathogenic fungus Metarhizium anisopliaeon adult Sunn pest (Eurygaster integriceps Puton.).

Sunn pest, Eurygaster integriceps Puton (Heteroptera: Scutelleridae), is one of the major wheat pest in Iran and neighboring countries. In order to control this pest, every year hundred of thousands hectares of wheat crop are sprayed with synthetic pesticides in Iran. Biocontrol, the use of living organisms as pest control agents, became an important alternative to the use of chemical pesticides and, therefore, a potential means of reducing pesticide use and its undesirable effects on human health and the environment. Especially, entomopathogenic fungi offer exciting possibilities as biocontrol agents because of their damaging effects on the target insect host and their ability to cause epizootics. So, in the current study, pathogencity of two isolates (M189, 4556) of Metarhiziurn anisopliae (Metschnikoff) with five concentrations (1x10(4), lx10(5), lx 10(6), 1x 10(7), 1x 10(8)) on adult Sunn pest was evaluated. Each treatment consisted of 30 adults that replicated three times. The adults were dipped in the conidia concentrations for about 10-15 seconds, then were taken and transfered to filter paper to drain off excess liquid. Controls were treated only with distilled water plus Tween 80 (0.02 % ). Treated adults were maintained in growth chamber (25+/-1 degrees C, RH 80+/-5 %) and mortality was recorded daily over two weeks. Probit analysis showed that LCso for M 189 and 4556 were 7.704x10(5) and 3.38x10(5), respectively. The results revealed that isolate 4556 was more effective on Sunn pest than the M189 isolate. Our results showed that different isolates of M. anisopliae have different effects on Sunn pest adult. So, identification, application and screening of different isolates in bioassays will provide a promising potential for finding of efficient isolate to be used in field studies as bio-pesticides.

Toxicity of fenitrothion, an organophosphorus pesticide, against summer population of sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae).

The sunn pest are a group of insect representing several genera of the shield bug (Scutelleridae) and stink bug (Pentatomidae) families, with the species Eurygaster integriceps being the most economically important. The sunn pest is a major pest of wheat and barley in central and western Asia and it accounts for annual losses of 20-30% for barley and up to 100% for wheat. In the recent decades problems have intensified largely due to change in farming practices. Thus this pest threatens food security and reduces the stability of traditionally wheat-based agricultural systems. In order to control pest, the major organophosphorus insecticide used is fenitrothion. So, the aim of the current study was to determine toxicity of fenitrothion against summer population of adult sunn pest. Insecticide toxicity was measured using a topical bioassay. To do so, the toxin was dissolved in acetone and six concentrations of toxin including 25, 50, 100, 150, 175 and 200 ppm were prepared. Insects were treated on the pronotum with one micro litre aliquots of insecticide in acetone (for treatments) or acetone alone (for control). The dose-mortality relationship was assessed from above mentioned doses with 30 insects treated per dose. After treatment, insects were maintained at laboratory conditions and mortality was recorded after 48 hours. Results were corrected for control mortality with Abbott's formula and then analyzed and toxicity determined. The obtained results showed that LD10, LD50, and LD90 were 34.7, 90.43, and 235.5, respectively.

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.

Esterase activity in summer population of sunn pest, Eurygaster integriceps Put. (Hemiptera: Scutelleridae).

A key constraint on increasing wheat production in Iran and some neighbouring countries is Sunn pest which cause severe damage to vegetative growth stage of plant in the early season. It also feeds on wheat grain in late growth stage of plants thus damaged wheat grains loose their bakery properties. Because of injecting protease enzymes into the grain during feeding, enzymes degrade gluten proteins and cause rapid relaxation of dough which results in the production of bread with poor volume and texture. Organophosphorus insecticides are the main pesticides used to control the insect pest. However, suitable reduction in pest population has not been achieved partly due to resistance to pesticides. Esterase plays crucial roles in insect physiology and detoxifies a broad range of xenobiotics including insecticides. Enhanced esterase activity is a major mechanism if insecticide resistance and has been detected in a number of insects. To evaluate esterase activity adult bugs were collected from wheat field in Karaj area of Iran and transferred to the laboratory. For biochemical assay, two adult bugs (either males or females) were homogenized in 500 microl Na-phosphate buffer pH 7.2. The homogenates were centrifuged at 14000 g for 10 minutes at 4 degrees C. The supernatants as the enzyme source were pooled and stored at -20 degrees C for later use. For enzyme assay, 300 microl of supernatant was mixed with equal volume of substrates (30 mM alpha-naphthyl acetate or 30 mM beta-naphthyl acetate) and incubated at 30 degrees C for 30 minutes. Then, 50 microl of fast blue solution (150 mg fast blue B in 15 ml distilled water plus 35 ml 5% SDS) was added and esterase activity was determined in a spectrophotometer at 595 nm. Data showed that there are no differences in esterase activity between male and female. However, There was significant differences between hydrolysis of substrates, alpha-naphthyl acetate and beta-naphthyl acetate. Insect esterase hydrolyzes alpha-naphthyl acetate much more than beta-naphthyl acetate.

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.

Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae).

The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases.

Effect of entomopathogenic fungus Tolypocladium species metabolite efrapeptin on Galleria mellonella agglutinin.

In the current study it has been found that efrapeptins, secondary metabolites of entomopathogenic fungi Tolypocladium cylindrosporum, interfere with agglutinin. The effect of efrapeptins on G. mellonella agglutinin was tested using rabbit blood. The results revealed that the end point for control larvae were 12.5 whereas for treated larvae which injected with 5 microl of 5 microg efrapepins ml(-1) or 50 microg efrapepins ml(-1) the end points were 10.5 and 8.5, respectively. Considering that efrapeptins suppress agglutination this study suggest that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes. It has been suggested that majority of interactions between cellular and humoral components of the insect immune system are receptor-mediated.

Effects of efrapeptin and destruxin, metabolites of entomogenous fungi, on the hydrolytic activity of a vacuolar type ATPase identified on the brush border membrane vesicles of Galleria mellonella midgut and on plant membrane bound hydrolytic enzymes.

The brush border membrane of the insect midgut is an initial site for interaction of insecticidal proteins. We have investigated the possibility that it may contain a target site for two insecticidal fungal toxins, destruxin and efrapeptin, both of which are ATPase inhibitors. We have studied the effects of the toxins on the hydrolytic activity of a vacuolar type ATPase (V-ATPase) that we have identified from Galleria mellonella midgut columnar cell brush border membrane vesicles (BBMV) by its cation and pH dependence, sensitivity to proton pump inhibitors and K(m) (0.49 mM ATP). Efrapeptin strongly inhibited the BBMV V-ATPase but destruxin had little effect. We compared the effects of the inhibitors on known plant membrane hydrolytic enzymes, and although the vacuolar pyrophosphatase and plasma membrane ATPase were not inhibited by the toxins, the V-ATPase from mung bean, but not barley, was inhibited (50%) by 10 microM concentrations of both compounds. Different forms of the toxins were tested on the ATPases and destruxin B and efrapeptin F were the most effective. Kinetic analysis showed that the purified forms of both compounds inhibited the V-ATPases uncompetitively and modelling of data for inhibition of the BBMV V-ATPase by efrapeptin at concentrations of 0.06--12 microM yielded a K(i) of 0.125 microM.