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A methanolic extract of Artemisia annua was obtained to evaluate its insecticidal activities against the sunn pest (Eurygaster intefriceps). Also, the responses of general esterase (EST), glutathione S-transferase (GST), alkaline phosphatase (ALP), acid phosphatase (ACP), acethylcholinesterase (AChE) to the plant extract were investigated. Topical application of plant extract on adults showed that the mortality was dose-dependent i.e. with increasing of plant extract concentrations more mortality achieved. Esterase and GST activities were increased in the first 24 h post-treatment. However, the enzymes activities were decreased after 24 h until 72 h. The activities of ALP, ACP and AChE in insect body decreased significantly and inhibition was higher along with increasing concentrations of plant extract. Isozyme electrophoresis profiles indicated that responses of isozymes (EST and GST) to plant extract were decreased after 48 h exposure to extract so that some enzymes bands disappeared. The results indicated that the highest concentration of A. annua extract was the most toxic among the four extracts. The decline of the detoxification ability in insects' tissues might be the main reason for the insecticidal activities.
Protein digestion in insects relies on several groups of proteases, among which trypsin plays a prominent role. In the current study, larvae of Pieris brassicae L. were fed radish leaves treated with 1 mM concentrations of three specific inhibitors of trypsin: AEBSF.HCl [4-(2- -aminoethyl)-benzenesulfonyl fluoride, monohydrochloride], TLCK (N-a-tosyl-l-lysine chloromethyl ketone) and SBTI (Soybean Trypsin Inhibitor) to find their potential effects on gene expression of trypsin. Initially, RT-PCR analysis revealed a gene of 748 bp responsible for synthesizing the digestive trypsin in P. brassicae larvae. Also, qRT-PCR data indicated a statistically greater expression of trypsin gene in the larvae fed 1 mM concentrations of AEBSF.HCl, TLCK and SBTI than the control. Results of the current study indicated that synthetic inhibitors can not only negatively affect the gene expression of P. brassicae trypsin, but also the insect can activate a compensatory mechanism against interruption of protein digestion by inducing more expression of the gene and producing more trypsin into the midgut lumen.
The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel. Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.
The striped rice-stem borer, Chilo suppressalis (Walker) is one of the most important pest of rice worldwide. In this study, a proteinaceous extract from Citrullus colocynthis L. shows various degrees of inhibition to digestive α-amylases of C. suppressalis. Digestive α-amylases of larvae were inhibited by different concentrations (approximately 50%) of C. colocynthis amylase inhibitor (CCAI). One of the isozymes totally disappeared and the sharpness of another decreased on native-PAGE electrophoresis. The pH dependency of inhibition revealed that the enzyme was inhibited in a wide range of pH from 7-10, and the optimal pH of the enzyme occurred in lepidopteran larvae. The highest inhibition of α-amylase by CCAI was observed at 25°C; the temperature near the optimal temperature of amylolytic activity. A time-course experiment demonstrated that enzymatic activity was the highest, 30 min after the onset of the experiment, when the highest inhibition occurred. The enzyme kinetic studies using Lineweaver-Burk analysis, revealed a mixed inhibition of CCAI on α-amylase activity. The current study is the first basic one using α-amylase inhibitor against C. suppressalis. This study opens the way for transgenic rice varieties containing inhibitors.
Essential oil of Artemisia annua L. was investigated to find out its toxicity and physiological aspects on the slug Agriolimax agrestis, in controlled conditions (8±1°C, 75±5 RH and 14:10 LD). The slugs received different concentrations of essential oil treated radish leaves in methanol, while the control received methanol alone. LC10, LC30, LC50 and LC90 values were estimated at 4.67, 5.3, 5.81, 7.25%, respectively. The effect of the essential oil on some important enzymatic components like; cytochrome P450 monnooxygenase, acid phosphatase, alkaline phosphatase, lipase, amylase and protease were significantly increased compared to the control. These results indicate that the plant Artemisia annua L. not only shows toxicity but also shows some irreversible effect on some important biochemical components and deserves further investigation.
The effects of pyriproxyfen were determined on the cellular immunity and phenoloxidase activity in the 4th instar larvae of Chilo suppressalis Walker. The bioassay results revealed the effective concentrations of: 10L : 18C, 30L : 72C and 50L : 190C μg · ml–1. The sole effect of 18 and 72 μg · ml–1 concentrations at intervals of 1–3 h caused a higher number of total hemocytes in the treated larvae than the control, but the reverse results were observed after 6–24 h. The number of plasmatocytes was lower than that of the control for intervals of 3–24 h but the number of granulocytes was higher than the control after 1–3 h although no significant differences were observed at the other times. In the treated larvae, the activities of phenoloxidase were higher and lower than those of the control after 1–3 h and 6–24 h, respectively. The combined effects of pyriproxyfen and the entomopathogenic fungus, Beauveria bassiana isolate B3 caused higher numbers of total hemocytes, plasmatocytes, and granulocytes in the treated larvae by use of the three concentrations of pyriproxyfen, at intervals of 6 and 12 h. Although the numbers of nodules in the larvae treated with concentrations of 18 μg · ml–1 were higher than those of other treatments, the overall numbers were lower than those of the control. Finally, the activity of phenoloxidase in the treated larvae was higher than that of the control, at intervals of 6 and 12 h post-treatment. Findings of the current study indicate an intervening role of pyriproxyfen in the cellular immunity of C. suppressalis to entomopathogenic objects.
In the current study, the entomotoxic effects of a lectin, extracted from Polygonum persicaria, were determined on Pieris brassicae larvae. The purified molecule significantly decreased larval survival, weight, and mass protein. Different concentrations of P. persicaria agglutinin (PPA) (0.5, 1 and 2 mg/ml) significantly decreased nutritional indices including: the efficiency of conversion of ingested food (ECI), the efficiency of conversion of digested food (ECD), and approximate digestibility (AD) in a dose-dependent manner. There was a significant decrease in larval α-amylase activity after larval fed on PPA. The decreased activity was found in both the biochemical assessment and gel electrophoresis; sharpness of one isozyme decreased and another completely disappeared. Meanwhile, PPA significantly decreased activities of glucosidases mainly β-glucosidase. Fed of P. brassicae larvae on different concentrations of PPA, significantly decreased activity of TAG-lipase in both the biochemical assessment and gel electrophoresis. It was found that alkaline phosphatase (ALP) and acid phosphatase (ACP) were also affected by PPA but concentrations of 1 and 2 mg/ml showed statistical differences compared to the control. General and specific proteolytic activities statistically decreased after larvae fed on different concentrations of PPA. Results of our study could bring attention to this molecule, showing it to be a safe and efficient control of the pest worldwide.
Third-instar larvae of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) were exposed to 12.5, 25, and 50 mg/kg concentrations of cadmium (Cd). The third-instar larvae were also exposed to 25, 50, and 100 mg/kg concentration of copper (Cu), and 25, 50, and 100 mg/kg concentrations of zinc (Zn). The heavy metal concentrations were each introduced separately into the artificial diet of the third-instar larvae. The third-instar larvae were 24 h old at the start of the treatment. The larvae were maintained in controlled conditions (26±1°C, 65±10% RH and 16L : 8D h) for 7 days. The feeding indices and the level of total protein, glycogen, cholesterol, and triglyceride were measured after the treatments. The results showed that high concentrations of Cd significantly increased approximate digestibility (AD). The relative growth rate (RGR) was significantly enhanced with a 25 mg/kg concentration of Cu. Efficiency of conversion of the ingested food (ECI%) increased significantly with lower concentrations of copper (25 and 50 mg/kg). The amount of cholesterol was also enhanced with 12.5 and 25 mg/kg concentrations of cadmium while the amount of triglyceride was significantly reduced in all Cd treatments. Glycogen, protein, and cholesterol were significantly enhanced in all Cu treatments. The amount of triglycerides at 25 and 50 mg/kg of Cu was significantly increased. Glycogen in all Zn treatments was significantly increased. Protein and cholesterol levels showed significant reduction with a 25 and 50 mg/kg concentration of zinc while triglyceride was enhanced with a 50 mg/kg concentration of Zn. It is clear from the present results that the presence of such heavy metals in the environment has an intense impact on H. armigera as far as food consumption and biochemical indices are concerned. Therefore, a need is shown for a more comprehensive study on the life table and immunology of this insect, under the presence of heavy metals.
Five morphological types of hemocytes were recognized in hemolymph of the 4th instar larvae of Hyphantria cunea (Drury). These hemocytes were: prohemocytes, plasmotocytes, granulocytes, oenocytoids, and spherulocytes. Tests were done on the effects of four isolates of the entomopathogenic fungus Beauveria bassiana (Bals.-Criy) (Fashand, spt-22, Ir-K-40 and 566), one isolate of Isaria farinosae (Holmsk.) Fr. (1872c), and latex-beads on the cellular immune defense mechanism and Phenoloxidase (PO) activity of H. cunea. Observation showed that plasmatocytes and granulocytes engulfed fungal pathogens by phagocytosis. The most phagocytosis occurred 30 and 60 min after injection but nodulation occurred in 3 and 6 hours, in all treatments. The total hemocyte count (THC) and granulocyte, and plasmotocyte numbers increased after the injection of spores. Phenoloxidase activity was determined in the presence of L-DOPA (L-3,4-dihydroxyphenylalanine), as a substrate in intervals, after injection of fungal spores, and latex beads. These studies demonstrated that B. bassiana is a promising candidate for biological control of H. cunea.
Samples were prepared from the midgut of 4th instar larvae of the crimson speckled moth Utethesia pulchella L. to find proteolytic activity and properties. Result revealed the presence of high proteolytic activity in the midgut when taking into account specific proteinases including trypsin-like, chymotrypsin-like, elastase and two exopeptidase (aminopeptidase and carboxipeptidase). The optimal pH of general protease was 8 and 7 when using azocasein and hemoglobin as general substrates, respectively. The optimal temperature of the total proteolytic activity in the midgut of U. pulchella was 25°C and 30°C when using azocasein and hemoglobin, respectively. Proteolytic activity was inhibited significantly by soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), trypsin inhibitor (TLCK), chymotrypsin inhibitor (TPCK) and Phenanthroline. These results provide evidences for the presence of serine proteinases as the major proteases in the midgut of U. pulchella; a key rangeland pest in warm climates. The interaction between digestive proteases and protease inhibitors have potentially important consequences for pest management programs.
Development and demography of Adalia decempunctata L. were studied under laboratory conditions at seven constant temperatures (12, 16, 20, 24, 28, 32 and 36°C). First instar larvae failed to develop to second instar at 12°С and no development occurred at 36°C. The total developmental time varied from 47.92 days at 16°C to 15.94 days at 28°C and increased at 32°C. The lower temperature thresholds of 11.05 and 9.90°C, and thermal constants of 290.84 day-degree and 326.34 day-degree were estimated by traditional and Ikemoto- Takai linear models, respectively. The lower temperature threshold (Tmin) values estimated by Analytis, Briere-1, Briere-2 and Lactin-2 for total immature stages were 11.99, 12.24, 10.30 and 10.8°C, respectively. The estimated fastest developmental temperatures (Tfast) by the Analytis, Briere-1, Briere-2 and Lactin-2 for overall immature stages development of A. decempunctata were 31.5, 31.1, 30.7 and 31.7°C, respectively. Analytis, Briere-1, Briere-2 and Lactin-2 measured the upper temperature threshold (Tmax) at 33.14, 36.65, 32.75 and 32.61°C. The age-stage specific survival rate (sxj) curves clearly depicted the highest and lowest survival rates at 16 and 32°C for males and females. The age-specific fecundity (mx) curves revealed higher fecundity rate when fed A. gossypii at 24 and 28°C. The highest and lowest values of intrinsic rate of increase (r) were observed at 28 and 16°C (0.1945 d–1 and 0.0592 d–1, respectively). Also, the trend of changes in the finite rate of increase (λ) was analogous with intrinsic rate of increase. The longest and shortest mean generation time (T) was observed at 16 and 28°C, respectively and the highest net reproductive rates (R0) was estimated at 24 and 28°C. According to the results, the most suitable temperature seems to be 28°C due to the shortest developmental time, highest survival rate, and highest intrinsic rate of increase.
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