Chronic effect of copper on biology, immunity, and biochemical assessment of Helicoverpa armigera (Lepidoptera; Noctuidae) in laboratory bioassays.

Although copper is an essential element for any organism's well-being, it becomes toxic if present in excess. In the present study, copper was provisioned at 25, 50, and 75 mg/kg in an artificial diet and fed to juvenile larvae of cotton bollworm, Helicoverpa armigera (Lepidoptera; Noctuidae), for 4 generations. The results of this investigation exhibited shortening of larval life in the first 2 generations, but extended duration was observed in third and fourth generations compared to controls, and dietary copper caused reduced total hemocyte counts in all treatments. The number of immunocytes (i.e., granulocytes and plasmatocytes) were also significantly reduced. The changes in activities of certain important enzymes, including catalase, superoxide dismutase, and peroxidases, were seen. Furthermore, after treatment, an increase in the activity of 2 detoxifying enzymes, glutathione s-transferase and acetylcholinesterase, was observed. It is clear that metallothioneins are important in maintaining essential and nonessential metal ion homeostasis. While copper is typically regarded as an important essential metal in an organism's life, excessive amounts can have deteriorating effects. This heavy metal is being used as a nano-based pesticide. Therefore, the present investigation aims to determine the fate of Cu in insects receiving them in new formulations.

Efficiency of Artemisia annua L. essential oil and its chitosan/tripolyphosphate or zeolite encapsulated form in controlling Sitophilus oryzae L.

The rice weevil, Sitophilus oryzae L., is one of the most widespread and destructive stored-product pests and resistant to a wide range of chemical insecticides. In this research, Artemisia annua L. essential oil (EO) and its encapsulated form by chitosan/TPP (tripolyphosphate) and zeolite were tested against S. oryzae adults. The order of toxicity was chitosan/TPP (LC30: 30.83, LC50: 39.52, and LC90: 72.50 µL/L air) > pure EO (LC30: 35.75, LC50: 46.25, and LC90: 86.76 µL/L air) > EO loaded in the zeolite (LC30: 43.35, LC50: 55.07, and LC90: 98.80 µL/L air). These encapsulated samples were characterized by dynamic light scattering (DLS) and field emission scanning electron microscope (FE-SEM) which revealed the size and morphology of the droplets measuring 255.2 to 272 nm and 245 to 271.8 nm for EO loaded in chitosan and zeolite respectively. The encapsulation efficiency and loading percentages of A. annua EO in chitosan/TPP and zeolite were 40.16% and 6.01%, and 88% and 85%, respectively. Fumigant persistence was increased from 6 days for pure EO then, 20 and 22 days for encapsulated oil in zeolite and chitosan/TPP, respectively. Our results showed that A. annua EO contains (±)-camphor (29.29%), 1,8-cineole (12.56%), ß-caryophyllene (10.29%), α-pinene (8.68%), and artemisia ketone (8.48%) as its major composition. The activity level of glutathione S-transferase increased while general esterase and acetylcholinesterase activity were significantly inhibited in the treated group compared with the control. Antioxidant enzymes, including catalase, peroxidase, and superoxide dismutase were activated in treated adults compared to controls. The current results suggest that encapsulation of A. annua EO by chitosan/TPP and zeolite in addition to safety and environmentally friendly approach could increase its sustainability and therefore enhancing the efficiency in controlling S. oryzae in storage.

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.

Effect of thyme essential oil and its two components on toxicity and some physiological parameters in mulberry pyralid Glyphodes pyloalis Walker.

Extensive usage of synthetic pesticides has proved to be destructive to all living being and the resurgence of pest resistance. Compounds derived from certain plants are usually safer compared to chemical control of pest. The present study thus intended to use Thymus vulgaris essential oil (EO) and two of its derivatives including thymol and carvacrol in order to see their deleterious effects on Glyphodes pyloalis (Walker). We also studied the oil components. This pest has recently become a serious concern for the silk industry. Our results showed that the thyme EO contain several components including thymol (26.9%), ρ-Cymene (14.54%), linalool (13.39%) and carvacrol (5.7%). Our toxicity tests revealed an estimated LD50 values for thyme EO, thymol and carvacrol 2.82, 32.18 and 56.54 µg/larva, respectively. However, the thyme EO was more toxic than its two tested compounds. The activity of certain detoxifying enzymes such as α- and ß-esterase, glutathione S-transferase and cytochrome P450 were significantly inhibited by thymol-treated larvae compared to the control group. Similarly, the activity of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatases enzymes in thymol-treated larvae decreased while the activity of acid phosphatases increased. Our results suggest that thyme EO and its components have potential for the control of G. pyloalis larvae in mulberry orchards, where no synthetic chemicals are allowed.

The sweet wormwood essential oil and its two major constituents are promising for a safe control measure against fall webworm.

The fall webworm, Hyphantria cunea (Drury), is a harmful polyphagous global defoliator. The major chemical components of Artemisia annua essential oil (EO) was found to contain (±)-camphor (16.42%), 1,8-cineole (6.22%), α-pinene (6%), caryophyllene (5.19%), and α-selinene (5.17%). The highest toxicity was recorded for EO of A. annua (LD50 = 305.05 µg/larva), followed by (±)-camphor (LD50 = 465.03 µg/larva) and 1,8-cineole (LD50 = 573.49 µg/larva). The binary mixtures of compounds expressed a weaker activity compared to individuals. The (±)-camphor was found to be antagonistic to 1,8-cineole. The biochemical compounds of treated larvae were also determined. The activity level of alanin and aspartate aminotransferase decreased sharply while acid and alkaline phosphatase increased. Activity of lactate dehydrogenase was significantly higher than the control group at 24 h, but decreased significantly after 48 h in all treatments. The activity of esterases were decreased in the treated larvae. The glutathione S-transferase significantly increased in all time intervals. Overall the current results suggest that the sweet wormwood (A. annua) EO and its components could be a safe and environmentally friendly approach in possible control of fall webworm (H. cunea).

Hemocytic cell line from the moth Glyphodes pyloalis (Lepidoptera: Crambidae) response to essential oils from Artemisia annua (Asterales: Asteraceae).

Extensive usage of synthetic chemical pesticides has collateral effects in harming human and animal health and the environment and promoting the development of resistance in pests. The potential of plant compounds as bio insecticides has been described as a promising field of agricultural development. The present study involved the use of Artemisia annua essential oils to evaluate their cytotoxic activities against an established cell line of lesser mulberry pyralid. Five types of hemocytes were recognized (prohaemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes) in the primary cultures maintained in Ex-Cell media with 10% fetal bovine serum (FBS). Artemisia annua essential oils produced noticeable cytotoxicity against the insect cell lines. Applied at a concentration 500 ppm, oils extracted from the vegetative or flowering stages of A. annua produced 71% and 80% cell death, respectively. Nanoemulsions of EOs from the vegetative or flowering stages of A. annua killed 67 and 60% of the cells, respectively. This study has clearly shown significant bioactivities of A. annua secondary metabolites to insect cell lines.

Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker.

In the present study, the toxicity and physiological disorders of the essential oil isolated from Artemisia annua flowers were assessed against one of the main insect pests of mulberry, Glyphodes pyloalis Walker, announcing one of the safe and effective alternatives to synthetic pesticides. The LC50 (lethal concentration to kill 50% of tested insects) values of the oral and fumigant bioassays of A. annua essential oil were 1.204 % W/V and 3.343 µL/L air, respectively. The A. annua essential oil, rich in camphor, artemisia ketone, ß-selinene, pinocarvone, 1,8-cineole, and α-pinene, caused a significant reduction in digestive and detoxifying enzyme activity of G. pyloalis larvae. The contents of protein, glucose, and triglyceride were also reduced in the treated larvae by oral and fumigant treatments. The immune system in treated larvae was weakened after both oral and fumigation applications compared to the control groups. Histological studies on the midgut and ovaries showed that A. annua essential oil caused an obvious change in the distribution of the principal cells of tissues and reduction in yolk spheres in oocytes. Therefore, it is suggested that the essential oil from A. annua flowers, with wide-range bio-effects on G. pyloalis, be used as an available, safe, effective insecticide in the protection of mulberry.

Toxicity and deleterious effects of Artemisia annua essential oil extracts on mulberry pyralid (Glyphodes pyloalis).

Botanical extracts are an important source of bio-pesticides and are generally considered safe to the environment. Artemisia annua L, a medicinal plant, well known for its antimalarial potential, was evaluated as a source of a type of essential oil collected during vegetative growth stage against Glyphodes pyloalis Walker. The main chemical components of the essential oil at vegetative stage of Artemisia annua was analyzed by GC- MS and contained 1,8-cineole (18.68%), Camphor (11.4%), α-Pinene (9.3%) and 3-Carene (6.3%). The LC50 of this plant oil was estimated to be 0.652% W/V and 2.585 µL/L air incorporated orally and fumigation, respectively. The digestive enzymes such as α-amylases, Proteases, Lipases and α- and ß-glucosidases were considereably inhibited in treated larvae compared with controls. Similarly, the amount of protein, glucose, and triglyceride were decreased in the treated larvae by methods used. The lower hemocyte numbers, nodule formation and activity of phenoloxidases after injection of Beauveria bassiana and latex beads showed its EO effect on immunity. The anatomy of the larval midgut after treatment showed degeneration in digestive cells. Emerging adult's ovaries showed significant changes in the ovarian sheath and lack of yolk spheres. The present investigation in accordance with our previous studies may ultimately lead to a formulation in controlling this notorious pest especially in mulberry orchards where the use of conventional chemicals is restricted.

Developing an Ephestia kuehniella Hemocyte Cell Line to Assess the Bio-Insecticidal Potential of Microencapsulated Helicoverpa armigera Nucleopolyhedrovirus Against Cotton Bollworm (Lepidoptera: Noctuidae) Larva.

Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) (genus: Alphabaculovirus, incertae sedis: Baculoviridae) has been used to control Helicoverpa armigera (Hübner). A reproducible and susceptible cell line was prepared from the hemocytes of Ephestia kuehniella in Grace and Ex-Cell 420 media. The population doubling time of these cloned cell cultures during the logarithmic phase were about 2.3 and 3.7 d for Ex-Cell 420 and Grace's media, respectively. When 60% confluence occurred, cells were infected by viral inoculums. All biochemical compounds were significantly changed relevant to cellular metabolism due to HearNPV infection. In order to improve its stability, two polymer formulations were used, i.e., formulation A (sodium alginate, gelatin, starch, and molasses) and formulation B (cottonseed kernel extract, Bran, glycerol, boric acid, egg white, and sugar). Formulant A provided high photostability by exhibiting 83.2 ± 3% efficacy and 88.66 ± 2.1% original activities remaining after 72 h UV exposure. Percentage original activity remaining of unformulated HearNPV and formulated mixture of B was 38.66 ± 2.6% and 9.33 ± 1.3%, respectively, after 72 h UV-irradiation. The virulence of the HearNPV proliferated from the Ex-Cell medium was similar to the virulence of wild-type HearNPV with LC50 of 7.7×105 OBs/ml. Formulant A, revealed only 20.0 ± 1% reduction in efficacy while the unformulated virus and formulant B faced a reduction of 90.0 ± 3% and 64.0 ± 2% after 72 h of UVA irradiation. Formulant A thus showed a high potential to protect HearNPVs microparticles against UV-inactivation suggesting a new platform for more efficient biological-management of cotton bollworm (specific name Helicoverpa armigera, genus: Helicoverpa, Lepidoptera: Noctuidae) in vivo.

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.

Can Feeding of Silkworm on Different Mulberry Variety Affect Its Performance?

This study attempted to identify a suitable host plant for rearing hybrid 31*32 silkworms. Four varieties of mulberry leaves including Ichinose, Kenmochi, Kines, and local were supplied to hybrid 31*32 of silkworm and their performance was assessed. Variables measured included nutrition, biochemistry, and economic parameters of silkworm. Number of cocoons, individual and total cocoon weight, cocoon shell weight, and cocoon yield/10,000 per larvae were significantly greater on the Kines variety compared with the others. Similarly, nutritional indices including efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), relative growth rate (RGR), and consumption index (CI) were greater for the larvae fed on Kines. Digestive and antioxidant enzyme activity of the larvae differed with variety. Our results suggest that Kines could be considered as an appropriate host plant for rearing of silkworms based on the study parameters.

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.

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.

Efficacy of Nanoencapsulated Thymus eriocalyx and Thymus kotschyanus Essential Oils by a Mesoporous Material MCM-41 Against Tetranychus urticae (Acari: Tetranychidae).

Inspite of well-established potentiality of plant essential oils as biopesticides, their environmentally low persistence is considered as a hindering obstacle for its commercialization. In the present study, chemical composition and toxicity of essential oils isolated from leaves of Thymus eriocalyx and Thymus kotschyanus were evaluated against two-spotted spider mite, Tetranychus urticae. The chemicals present in the crude oil were found to be thymol (28.83%), oleic acid (11.51%), palmitic acid (8.60%), borneol (5.72%), ρ-cymene (3.60%), and 1,8-cineole (3.57%) in the essential oil of T. eriocalyx, and camphene (35.59%), linalyl acetate (20.47%), linalool (14.75%), α-terpineol (13.87%), and geranyl acetate (3.07%) in the essential oil of T. kotschyanus. The essential oils had strong fumigant toxicity on the adult females of Te. urticae and their fumigation persistence was prolonged until 6 and 5 d, respectively, for T. eriocalyx and T. kotschyanus. Loading of essential oils in MCM-41 increased their stability and persistence was extended up to 20 and 18 d for T. eriocalyx and T. kotschyanus. Further, mite mortality increased from 80 to 203 mites by T. eriocalyx and from 58 to 186 mites by T. kotschyanus nanoencapsulated essential oils. Based on these results, nanoencapsulation of T. eriocalyx and T. kotschyanus essential oils in MCM-41 may be a useful method for their application in the management of Te. urticae.

Acaricidal Potentials of the Terpene-rich Essential Oils of Two Iranian Eucalyptus Species against Tetranychus urticae Koch.

There is a rapid growth in the screening of plant materials for finding new bio-pesticides. In the present study, the essential oils of E. oleosa and E. torquata leaves were extracted using a Clevenger apparatus and their chemical profiles were investigated by Gas Chromatography-Mass Spectrometry (GC-MS). Among identified compounds, the terpenes had highest amount for both essential oils; 93.59% for E. oleosa and 97.69% for E. torquata. 1,8-Cineole (31.96%), α-pinene (15.25%) and trans-anethole (7.32%) in the essential oil of E. oleosa and 1,8-cineole (28.57%), α-pinene (15.74%) and globulol (13.11%) in the E. torquata essential oil were identified as the main components. The acaricidal activity of the essential oils of E. oleosa and E. torquata were examined using fumigation methods against the adult females of Tetranychus urticae Koch. The essential oils have potential acaricidal effects on T. urticae. The essential oil of E. oleosa with LC50 value of 2.42 µL/L air was stronger than E. torquata. A correlation between log concentration and mite mortality has been observed. Based on the results of present study, it can be stated that the essential oils of E. oleosa and E. torquata have a worthy potential in the management of T. urticae.

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.

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.

External morphology and calling song characteristics in Tibicen plebejus (Hemiptera: Cicadidae).

Tibicen plebejus is the largest cicada native to the ecosystem in northern Iran. The male cicada produces a loud calling song for attracting females from a long distance. It is presumed that the female selects a mate based on a combination of passive and active mechanisms, but it is not known if she selects for size, nor if the male's size correlates with any characteristic of the advertisement call. In this study, we report the relationship between calling song features and morphological characters in the male of T. plebejus. Research was conducted in northern Iran during the summer of 2010. Seventeen males were collected and their calling songs were recorded in a natural environment. Two morphological characters were measured: length and weight. Maximum, minimum and average of values of 10 key acoustic variables of the calling song were analyzed: phrase duration, phrase part 1, phrase part 2, number of phrases per minute, echeme duration, echeme period, interecheme interval, number of echeme per second, echeme/intereheme ratio, and dominant frequency. The data were tested for the level of association between morphology and acoustic variables using simple linear regression. In conclusion, in terms of song structure, three significant positive correlations existed between length and (1) mean echeme duration, (2) mean echeme/interecheme ratio, (3) maximum echeme/interecheme ratio. We found out also four significant negative correlations between both length and weight with (1) minimum interecheme intervals, (2) mean dominant frequency, (3) minimum dominant frequency, (4) maximum dominant frequency, and between weight and (1) minimum interecheme intervals, (2) mean dominant frequency, (3) minimum dominant frequency, (4) maximum dominant frequency. It can be found that larger males of T. plebejus produce songs of lower frequency and are less silent between echemes.

A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 µmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

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.