Morpho-molecular study of entomopathogenic fungi associated with citrus orchard pests in Northern Iran.

Entomopathogenic fungi play a significant role in regulating insect populations in nature and have potential applications in pest management strategies in different regions. Citrus spp. are among the important horticultural products in northern Iran, and the orchards are affected by different insect pests, especially mealybugs. This study aimed to isolate and identify entomopathogenic fungi associated with citrus orchard pests in northern Iran, focusing on Akanthomyces and Lecanicillium species on mealybugs. Through the samples collected from different regions within Guilan province, 12 fungal isolates were collected and identified based on the combination of morphological characteristics and molecular data. Akanthomyces lecanii, A. muscarius, Engyodontium rectidentatum, Lecanicillium aphanocladii and Lecanicillium rasoulzarei sp. nov. were identified. Of these, A. muscarius on Lepidosaphes sp., E. rectidentatum on Coccidae, and L. aphanocladii on Tetranychus urticae are reported as new fungal-host records from Iran. Moreover, a new species, Lecanicillium rasoulzarei, is illustrated, described, and compared with closely related species.

Toxicity and physiological interruptions of a proteinaceous toxin from Metarhizium anisopliae against the olive fruit pest, Bacterocera oleae (Diptera: Tephritidae).

The use of natural bioactive compounds mainly proteinaceous secondary metabolites of fungi is one of the promising pest control methods because of their lethal effects on insects in low concentration, limited persistence in environment and easily decomposition into environmentally safe compounds. The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a destructive pest of olive fruits around the world. In the current study, the proteinaceous compounds were extracted from the two isolates (MASA and MAAI) of Metarhizium anisopliae and their effects were evaluated on toxicity, feeding performance and antioxidant system of the adult's olive flies. Both extracts from MASA and MAAI showed entomotoxicity against the adults by 2.47 and 2.38 mg/mL as LC50 concentrations. Also, LT50 values were recorded 1.15 and 1.31 days for MASA and MAAI, respectively. No statistical differences were recorded in the consumption rate of the adults on control and secondary metabolite contained protein hydrolysate. In contrast, the adults fed on LC30 and LC50 concentrations of MASA and MAAI demonstrated significant reduction in the activities of digestive alpha-amylase, glucosidases, lipase, trypsin, chymotrypsin, elastase, amino- and carboxypeptidases. Activity of antioxidant enzymes changed in the adults of B. oleae fed on the fungal secondary metabolites. Catalase, Peroxidase and Superoxide dismutase elevated in the treated adults with the highest amounts of MAAI. Similar results were found in activity of ascorbate peroxidase and glucose-6-phosphate dehydrogenase except for malondialdehyde amount in which no statistical differences were recorded between treatments and control. Relative gene expression of caspase enzymes revealed the higher expression in the treated B. oleae compared to control with the highest level of caspase 8 for MASA and caspases 1 and 8 for MAAI. Results of our study showed that the secondary metabolites extracted from the two isolates of M. anisopliae caused mortality, interrupted digestion and induced oxidative stress in the adults of B. oleae.

Effect of cold storage on some biological and physiological performance of Adalia decempunctata L.

Adalia decempunctata (L.) is a common predator in agro ecosystems. Here, the effects of low-temperature storage on biological parameters, energy reserves, and antioxidant enzymes were investigated. Eggs, larvae, and pupae of this predator were maintained for 1-4 weeks and adults for different days (20, 35, 50, and 65 d) at 4, 7, and 10 °C in darkness and 70 ± 5% R.H. After storage of 20 days, the survival rate in male and female insects was >90% in 4, 7, and 10 ± 1 °C. In 65 days, the highest survival rate for adult insects was recorded at 4 °C. Effect of cold storage on the performance of adult's A. decempunctata was not statistically different for periods up to 35 days, but decreased longer duration (65 days). The female adults were able to tolerate the cold storage period by producing higher amounts of glucose and antioxidant enzymes, thus displaying a high survival rate. Based on our results, maintaining A. decempunctata adults at low temperatures (i.e. 4, 7, and 10 °C) for 20 or 35 days performed best without loss of quality.

Changes in immune responses, gene expression, and life table parameters of Helicoverpa armigera Hübner fed on a diet containing the saponin of tea plant, Camellia sinensis.

Saponins cause mortality in insects by reducing food absorption and movement in the gut, which may be used to exploit the control of insect pests. In the current study, tea seed saponin (TSS) was extracted and then added to the artificial diets of Helicoverpa armigera. Pre-ovipositional period of the TSS-treated individuals increased while longevity and fecundity decreased compared to control. There was a significant reduction of the treated individuals in the life table parameters of TSS-treated Individuals including net reproduction rate (R0 ), intrinsic rate of population increase (r), finite rate of increase (λ), and gross reproduction rate (GRR). Also, we found that saponin suppressed the immune system by reducing the total hemocyte count, immune-related gene expression, and phenoloxidase activity. Our results demonstrated a lower expression of cecropin gene in the treated larvae with TSS while no significant differences were observed in attacin gene. Our results clearly showed that feeding of H. armigera larvae in the diet containing TSS significantly reduced demographic parameters, forced insects to obtain more time to complete one generation, and caused vulnerabilities against pathogens. These discrepancies alleviated nutrient uptake of the larvae and disrupted their feeding and growth. Hence, a proper formulation with a desirable concentration would be prepared and applied in the fields suffering H. armigera damage to monitor insecticidal efficiency of TSS.

Toxicity, antifeedant and physiological effects of trans-anethole against Hyphantria cunea Drury (Lep: Arctiidae).

Plant secondary metabolites are currently known to interfere with basic metabolic, behavioral and physiological processes of insects. In the current study, the biological and physiological effects of trans-anethole were investigated against Hyphantria cunea Drury. The bioassay data demonstrated the high toxicity of trans-anethole against the fourth-instar larvae with the LC30, LC50 and LC90 values of 0.72, 1.41 and 7.20 µL/mL, respectively. Also the concentrations of LC30 and LC50 showed 53 and 87% feeding deterrency against the larvae. The biochemical experiments revealed that oral exposure of trans-anethole decreased the activities of digestive enzymes, acetylcholinesterase and the contents of energy reserves while, it induced the activities of detoxifying and antioxidant enzymes compared to control. In fact, trans-anethole induced the inhibition of digestion and AChE activities accompanied by imbalance in metabolic and oxidative processes so it may be recommended as a potent biopesticide in control of H. cunea populations.

Immunological interactions of Chilo suppressalis Walker (Lepidoptera: Crambidae) with the native entomopathogenic fungi.

Entomopathogenic fungi can attack many insect hosts and have been applied as the eco-friendly alternatives to synthetic chemicals for the control of pests. Insects have developed different defense systems encountering entomopathogens including humoral and cellular immune responses. In the present study, injection of some native entomopathogenic fungi to the Chilo suppressalis Walker larvae resulted in an enhancement of the cellular and antimicrobial defenses. The numbers of total and differential hemocytes increased rapidly in the first 3 and 6 h but those gradually reduced 12 and 24 h post-injections. The nodule formation and phenoloxidase activity increased at the time intervals after fungal infection. A similar trend was found in the transcription of antimicrobial peptides including attacin1 and 2, cecropin1 and 2, gallerimycin, defensin, lysozyme, and prophenoloxidase-activating proteinase-3 during infection fungi. In all cases, the target gene transcription was upper in the larvae injected by the fungi than that of control larvae. These results may elucidate better knowledge on the interaction of the fungi present in agroecosystems with the target insect pest.

Screening and Virulence of the Entomopathogenic Fungi Associated with Chilo suppressalis Walker.

The current study aimed to explore the entomopathogenic fungi associated with the larvae of Chilo suppressalis Walker, a serious pest of rice, in northern Iran. The collected specimens were cultured and identified through morphological and molecular methods. The 38 specimens were identified by microscopic examination and genetic sequencing of the ITS region as follows: twenty-one isolates of Beauveria bassiana, five isolates of Akanthomyces lecanii, four isolates of Akanthomyces muscarious, three isolates of Metarhizium anisopliae, two isolates of Hirsutella subulata, two isolates of Trichoderma sp. and one isolate of Aspergillus sp. All the identified isolates were treated on the larvae through bioassay, evaluating the amount of hydrophobin and the activities of proteases, chitinases and lipase to find their virulence. Moreover, the percentage of thermotolerant and cold activity of the isolates were tested to determine their environmental persistence. The overall results revealed the isolates of B. bassiana, including BBRR1, BBAL1 and BBLN1 as the most virulent and environmental adaptive isolates among the fungi associated with C. suppressalis.

Methanolic Extract of Winter Cherry Causes Morpho-Histological and Immunological Ailments in Mulberry Pyralid Glyphodes pyloalis.

The effect of Withania somnifera a medicinal plant seed extract was tested against lesser mulberry pyralid, a potential pest of mulberry. The mulberry leaves were used for silk production in rural areas of northern Iran. The extract was administered orally by leaf dipping method in two lower (5%W/V) and higher (15%W/V) dosages to third instar larvae (<24 h) for biological assays and to fifth instar larvae (<24 h) for Physiological studies. The results showed formation of larvoids (Ls), larval-pupal intermediates (LPIs), pupoids (Ps) and pupal-adult intermediates (PAIs). The results showed increased larval duration by 1.7 and 2 folds in 5 and 15% treatment, respectively. Fecundity of resultant adults was decreased by 1.2 and 1.3 in 5 and 15% treatment, respectively. Except approximate digestibility (AD) and consumption index (CI) all other feeding indices showed reduction. The feeding deterrence was prominent at 15% (87%) and 5% showing 48% deterrence. Our enzymatic and non-enzymatic assessments upon treatment showed reduction in key components, except detoxifying enzymes. However, the activity of an important enzyme involved in cuticle hardening and immunity called phenoloxidase was reduced. We also investigated the histology of midgut for further analysis and found drastic changes in main cellular elements. Immunological changes following treatment was noticeable in reduced Total Hemocyte Count but surprisingly increased Differential Hemocyte Count. However, the hemocytes structure was extremely damaged. The reduced number of eggs in treated but survived adults indicated reduced ovaries, with vacuolization both in trophocytes and oocytes. The key chemical compounds showed reductions particularly at 15%. The present results are concomitant with few earlier studies on this medicinal plant and deserve further studies particularly in deriving key chemicals that alter metamorphosis similar to insect growth regulators.

Toxicity and physiological effects of ajwain (Carum copticum, Apiaceae) essential oil and its major constituents against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

The effects of Ajwain essential oil and its major constituents were investigated against newly fourth instar larvae of Tuta absoluta, a major pest of tomato cropping in Iran and other parts of the world. The study included individual efficacy of each compound and determination of their synergistic or antagonistic efficacy. We also determined the activities of detoxifying enzymes in treated insects. According to the results, the Ajwain essential oil and thymol showed the highest toxicity against T. absoluta larvae (LC50 = 6.14 and 7.72 µL/mL, respectively, and LC30 = 3.05 and 3.57 µL/mL, respectively). The ɤ-terpinene and p-cymene had lesser toxicity compared to other compounds. The larvicidal potency increased by binary mixtures of active constituents, such as thymol + p-cymene and thymol + É¤-terpinene. On the other hand, ɤ-terpinene + p-cymene mixture demonstrated antagonistic effect on the larvae. The esterases and glutathione S-transferases were significantly increased in all treatments. Finally, our results revealed a significant inhibition of AChE activity in the treated larvae by all treatments except for ɤ-terpinene + p-cymene. In vitro experiments representing AChE inhibition with IC50 values were recorded 0.370, 0.457, 0.528, 1.094 and 1.323 µL/mL for thymol + p-cymene, thymol + É¤-terpinene, thymol, ɤ-terpinene and p-cymene, respectively. Thymol demonstrated significant potential as a controlling agent of T. absoluta larvae, both larvicidal, compatible with other compounds with strong AChE inhibition properties. These finding could pave the way for development of new botanicals based on EO constituents which should be completed with preparation of an efficient formulation and field trials.

Alterations in some physiological processes of Bactrocera oleae Rossi (Diptera: Tephritidae) following pyridalyl treatment.

Pyridalyl is one of the recently discovered insecticides which has shown toxicity against several insect pests with slight- or non-lethal effects against natural enemies. Cytotoxicity and prevention of protein synthesis have been reported as pyridalyl mode of action. In the current study, adults of Bacterocera oleae, as a major pest of olive, were fed on protein hydrolysate diet containing LC50 concentration of pyridalyl to determine the possible mode of action through gene expression and amino acid profile. Results showed that treated flies had significantly higher gene expressions of cytochrome P450, insulin receptor-like, antioxidant enzymes (catalase, peroxidase, superoxide dismutase) and the enzymes involved in cell death; caspase1, caspase3, caspase8 and caspase9 compared to control. Moreover, determination of amino acid profile in the whole body of both control and pyridalyl-treated B. oleae revealed the significant higher amounts of glutamic acid, glutamine, histidine, threonine, citrulin, arginine, taurine, alanine, tyrosine, α-amino butric acid, methionine and isoleucine in the treated adults while the control flies had the higher amounts of serine, glycine and tryptophane. No significant differences were found in the amounts of asparagine and phenylalanine between control and treatments. These results are in accordance with earlier findings on mode of action of pyridalyl through activation of cytochrome P450 system and alterations in protein synthesis machinery although here other mechanisms were also reported including activation of insulin receptor and antioxidant enzymes. Moreover, it is the first report on cytotoxicity through apoptosis with engagement of caspases.

Effect of thermal stress on the immune responses of Chilo suppressalis walker (Lepidoptera: Crambidae) to Beauveria bassiana.

Temperature is one of the important environmental elements affecting ecological fitness of insects through alterations in physiological systems. In the current study, a comparison was made on the cellular and humoral immune responses of the Chilo suppressalis larvae exposed to thermal stress (34 °C) and optimal rearing temperature (24 °C). Although total hemocyte count increased in the injected larvae by Beauveria bassiana, elevation of hemocyte numbers was significantly different in the larvae exposed to 34 °C for a short-time period compared to long-term exposure and control. A similar trend was observed in plasmatocyte and granulocyte counts as well as phenoloxidase activity. Gene expression of some antimicrobial peptides, including attacin1, attacin2, cecropin1, cecropin2, defensin, gallerimycin, lysozyme and prophenoloxidase-activating proteinase-3, was compared in the larvae exposed to thermal regimes and injection challenges. In all cases, expression of the target genes was relatively higher in the larvae injected by B. bassiana and short-term exposure at 34 °C. The present results confirmed that C. suppressalis could modulate the immune system in response to different thermal stress conditions mainly over a short period.

Changes in immune responses of Helicoverpa armigera Hübner followed by feeding on Knotgrass, Polygonum persicaria agglutinin.

There is no study implying the effect of plant lectins on insect immune elements in both challenged and non-challenged conditions with entomopathogenic agents. Lectins may bind to immune receptors on the surface of insect hemocytes, thus inducing or even disabling common immune functions including hemocyte counts, nodulation/encapsulation, phenoloxidase activity, and synthesis of antimicrobial peptides. In the present study, effect of Polygonum persicaria L. agglutinin (PPA) on immune responses of Helicoverpa armigera Hübner was investigated by feeding artificial diet treated to the larvae. Subsequently hemocyte count and expression of some immune-related genes were considered for analyses. The two groups of larvae including control and PPA-treated (1%) were divided into four subgroups of intact, Tween-80 injected, latex-bead injected and Beauveria bassiana-injected. Except for intact larvae, the highest numbers of total and differential hemocyte counts were recorded 12 hr postinjection, however, the PPA-fed larvae showed a significantly lower hemocyte counts compared to control. The number of nodules in PPA-fed larvae was significantly lower than control, but the injected larvae of both control and PPA showed the highest nodulation 24 hr postinjection. Although the highest activity of phenoloxidase was observed 12 and 24 hr postinjection but its activity significantly decreased in PPA-fed larvae compared to control. Gene expression of antimicrobial peptides including attacin, cecropin, and peptidoglycan receptor proteins were significantly decreased in artificial diet-fed larvae containing PPA and then injected by B. bassiana spores and latex bead compared to control. These results clearly indicate adverse effects of PPA on immune responses in H. armigera.

Effects of α-pinene, trans-anethole, and thymol as the essential oil constituents on antioxidant system and acetylcholine esterase of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae).

The current study aimed to determine the potential effects of three essential oil constituents, α-pinene, trans-anethole, and thymol, on antioxidant system and acetylcholine esterase (AChE) of Ephestia kuehniella Zeller. The 4th instar larvae were initially fed on an artificial diet containing an LC50 concentration of each above-mentioned compounds separately prior to being undertaken for sample preparation and biochemical assays. The significant higher activities of superoxide dismutase, peroxidase and catalase were observed in the treated-larvae at both time intervals. Similar findings were found in the activity of glutathione S-transferase by using both reagents. Although activities of ascorbate peroxidase and glycerol-6-phosphate dehydrogenase increased in the treated larvae by all constituents while glycerol-6-phosphate dehydrogenase showed no statistically different activity among the larvae fed on α-pinene, trans-anethole, and thymol. The concentration of malondialdehyde and the ratio of oxidized (RSSR) to reduced (RSH) thiols showed statistical differences among control and treated larvae except for time interval of 24 h regarding the ratio of RSSR/RSH. Finally, our results demonstrated a significant decrease of AChE activity in the treated larvae by all constituents after 24 h while no statistical differences were found between control and trans-anethole after 24 h. Also, in vitro analysis revealed significant inhibition of AChE representing IC50 values of 0.864, 0.490 and 0.137 µl/ml for α-pinene, trans-anethole, and thymol, respectively. These results determined significant effects of administered constituents on induction of antioxidant system and inhibition of a nervous system component which expand our knowledge on physiological turbulences due to essential oil treatment.

A Laboratory Study on the Modeling of Temperature-Dependent Development and Antioxidant System of Chilo suppressalis (Lepidoptera: Crambidae).

The developmental rates of Chilo suppressalis (Walker; Lepidoptera: Crambidae) were investigated at different constant temperatures of 11, 18, 22, 24, 26, 30, 32, 34, and 36°C to find out temperature thresholds, thermal requirements and larval antioxidant responses. The time to complete immature stages separately reduced by raising the rearing temperature except for eggs which showed no significant differences at 30-34°C. Data analysis by traditional and Ikemoto-Takai linear models determined Tmin of 10.0 and 9.92°C as well as thermal constants of 840.34 and 848.0 DD for the overall immature stages. The models including Analytis, Briere-2, Lactin-2, and Sharpe-Schoolfield-Ikemoto (SSI) calculated Tmin values of 9.15, 9.57, 10.0, and 11.37°C for overall immature stages while Tfast was found to be 33.8, 33.3, 33.7, and 33.0°C, respectively. Tmax was calculated as 35.12, 34.66, 34.56, 36.84, 34.11, and 35.15°C for Analytis, Briere-2, Lactin-2, SSI, Logan-6, and Logan-10. Topt using SSI was calculated as 24.42°C for total developmental time. The larvae exposure to 34°C in the short-term period demonstrated the highest activities of catalase, peroxidase, and superoxide dismutase compared to control (24°C). Activities of ascorbate peroxidase and glucose-6-phosphate hydrogenase also increased at 34°C in the short-term period for all preparations. The larvae exposed to 34°C in short-term period showed the highest amounts of Malondialdehyde and oxidized and reduced thiols (RSSR/RSH) ratio compared to control. These results may be useful to explain potentially ecological performance of C. suppressalis as the major pest of rice in Iran.

Effect of Polygonum persicaria (Polygonales: Polygonaceae) Extracted Agglutinin on Life Table and Antioxidant Responses in Helicoverpa armigera (Lepidoptera: Noctuidae) Larvae.

Plant lectins could reduce insect populations by imposing imbalances in biology and physiology. Here, an agglutinin was extracted from Polygonum persicaria L. (PPA; Polygonales: Polygonaceae) and its effects were investigated on life table parameters and antioxidant system of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). PPA significantly changed demographic parameters showing adverse effects on age-stage survival rate (Sxj), age-specific survival rate (lx), age-specific fecundity rate (mx), age stage specific fecundity (fxj), and life expectancy (exj). Also, life table parameters including net reproduction rate (R0) (Offspring/female), intrinsic rate of population increase (rm) (days-1), finite rate of increase (λ) (days-1), gross reproduction rate (GRR) (Offspring/female) significantly decreased in the PPA-treated H. armigera compared to control except for mean generation time (T) (days). Activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CA), peroxidase (POX), glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (GPDH) increased statistically in the PPA-treated larvae compared to control while no significant difference was observed in the activity of ascorbate peroxidase (APOX) activity. Moreover, ratio of RSSR/RSH and concentration of malondialdehyde (MDA) were found to be statistically higher in PPA-treated larvae than control. The current results clearly showed that PPA not only had a negative impact on demography of H. armigera but also induced antioxidant raise by releasing free radicals. These released radicals, together with impaired digestion and absorption observed in our previous report, could be considered as a reason for reducing biological fitness of H. armigera.

Modeling Temperature-Dependent Development of Glyphodes pyloalis (Lepidoptera: Pyralidae).

Development of Glyphodes pyloalis Walker was studied under laboratory conditions at constant temperatures of 12, 16, 20, 24, 28, 30, 32, and 36 °C. No development occurred at 36 °C. Although eggs hatched at 12 ºC, no larvae were capable of developing to adult stage. At 16 ºC, survival rate was low (4%) and prepupal stage lasted 101.68 ± 11.03 d. Larvae completed development through six stadia at 16, 30, and 32 °C. Developmental time of overall immature stages varied from 46.62 d at 20 °C to 22.04 d at 30 °C and increased at 32 °C. The lower temperature thresholds of 10.30 and 11.22 °C, and thermal constants of 429.18 and 401.88 DD were estimated by traditional and Ikemoto-Takai linear models, respectively. The Tmin values estimated by Analytis, Briere-2, Lactin-2, and Sharpe-Schoolfield-Ikemoto (SSI) for overall immature stages were 12.40, 12.92, 9.00, and 13.04 °C, respectively. The fastest development temperatures (Tfast) of 31.1, 31.1, 30.8, and 30.7 °C were estimated for overall immature stages based on Analytis, Briere-2, Lactin-2, and SSI, respectively. The intrinsic optimum temperature (Topt) estimated from the thermodynamic SSI model for total developmental time was 24.63 °C, in which the maximal active state enzymes involved in developmental process. The nonlinear models of Analytis, Lactin-2, Briere-2, and SSI estimated the upper temperature thresholds (Tmax) at 36.66, 35.97, 38.88, and 34.05 °C, respectively. These findings could be used to predict the population dynamics of G. pyloalis for an effective management.

Effects of the Agglutinins Extracted From Rhizoctonia solani (Cantharellales: Ceratobasidiaceae) on Pieris brassicae (Lepidoptera: Pieridae).

Lectins are widespread proteins found in plants, fungi, bacteria, and vertebrates, and they play the critical roles in many physiological functions. Two lectin molecules (namely, RSAI and RSAII) were extracted from Rhizoctonia solani Kuhn and their effects on Pieris brassicae L. larvae were determined by larval survival rate, body mass, nutritional indices, digestive enzyme activities, and caspase-3 gene expression. The highest mortality caused by RSA treatment was recorded up to 80%, the larval weight decreased to 0.05 g and Similarly, RSAs significantly decreased nutritional indices including conversion efficiency of ingested food (ECI), conversion efficiency of digested food (ECD), approximate digestibility (AD), relative consumption rate (RCR), and relative growth rate (RGR) in a dose-dependent manner. Activities of α-amylase and α- and ß-glucosidases significantly decreased in the larvae fed with RSA-treated diets. Also, activities of TAG-lipase and proteases significantly reduced after feeding with different concentrations of RSAs. Gene expression analysis of caspase-3 in control and treated larvae revealed significant increment of its expression in the larvae fed with RSAI and RSAII, respectively, 9.52- and 1.47-fold compared to control. These results clearly demonstrated insecticidal effects of R. solani lectins on P. brassicae via several physiological pathways, thus rendering RSA as a good target for furthering our knowledge and suggesting new strategies to overcome pesticide side effects.

Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae).

The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had V max values of 4.64 and 3.02 U/mg protein and K m values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (ß-aminoethylether) N, N, N', N'-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.

Immune and metabolic responses of Chilo suppressalis Walker (Lepidoptera: Crambidae) larvae to an insect growth regulator, hexaflumuron.

Efficient control of Chilo suppressalis Walker is always controversial due to highly economic damage, resistance to insecticides and environmental pollutions. So, combination of safe pest controls e.g. biocontrol agents and insect growth regulators seems to be promising via integrated pest management program. Bioassay of hexaflumuron on 4th larval instars revealed concentrations of 44.34, 179.74 and 474.94µg/ml as LC10-50 values. Numbers of total hemocytes, plasmatocytes and granulocytes as well as phenoloxidase activity increased in the different time intervals following treatment by hexaflumuron. Combined effects of hexaflumuron and Beauveria bassiana Vuillemin also increased hemocyte numbers and phenoloxidase activity at different time intervals using all concentrations. Activities of general esterases assayed by α- and ß-naphtyl acetate and glutathione S-transferase using CDNB and DCNB increased 1-12h post-treatment. Activities of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase and aldolase increased in the larvae treated by hexaflumuron. However enhanced activity of lactate dehydrogenase was only obtained by treating 180 and 470µg/ml concentrations of hexaflumuron. Activities of ACP and ALP were found to be higher than control for all time intervals even 1-12h post-treatment. The amounts of HDL and LDL increased in the highest concentrations of hexaflumuron after 12-24h of post-treatment. Amount of triglyceride was higher than that of control after 1 and 3h but it was lower in other time intervals. Amounts of glycogen and protein were lower than those of control for all time intervals except for 6 and 12h of post-treatment in case of protein. Results of the current study revealed negative effects of hexaflumuron on intermediary metabolism of Chilo suppressalis but it increased the number of hemocytes and activity of phenoloxidase which are responsible for spore removal from hemolymph. It can be concluded that hexaflumuron is able to decrease survival and biological performance of C. suppressalis via intervening in intermediary metabolism but the given results showed incompatibility of the IGR with possible microbial control.

Effects of Potato Cultivars on Some Physiological Processes of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

Colorado potato beetle, Leptinotarsa decemlineata (Say), is an important pest of potato throughout the world. Here, the effects of six potato cultivars including 'Arinda,' 'Sprit,' 'Markiez,' 'Lotta,' 'Santae,' and 'Agria' were studied on nutritional indices, digestive enzymes, and some components involved in intermediary metabolism of L. decemlineata. Nutritional indices of the larvae and the adults were significantly different followed by feeding on various potato cultivars. The individuals fed on Agria showed the highest activity of digestive proteases although cathepsin B demonstrated same Activity on Santae and Lotta. The highest activity of α-amylase was found in the larvae fed on Arinda, but the adults demonstrated the highest amylolytic activities on Santae and Agria. Both larvae and adults of L. decemlineata fed on Santae revealed the highest α- and ß-glucosidase activities. No significant differences were found in lipase activity of larvae, but the highest lipase activity was found in the adults fed on Santae. The highest activities of transaminases were found in the larvae and adults fed on the Agria except for γ-glutamyl transferase. In case of aldolase, the highest activities were observed in the larvae and adults fed on Santae and Sprit. The highest activities of lactate dehydrogenase were obtained in the larvae and adults fed on Santae. The highest amount of low-density lipophorin was measured in both individuals fed on Santae. There were no significant differences in high-density lipophorin amount of adults, but the highest value was found in the larvae fed on Agria. The lowest amounts of protein and triglyceride were observed in both individuals fed on Santae and Agria, respectively. These results revealed Santae is the most suitable cultivar for L. decemlineata based on digestion and intermediary metabolism findings, but Lotta is an unsuitable cultivar and could be considered for integrated pest management.