Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab.

An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC₅₀) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed the highest insecticidal activity at 6 μM concentration within 48 h. The nutritional indices were also significantly affected by the 6 μM concentration (P < 0.05). Food consumption, efficiency of conversion of ingested and digested food, relative growth rate, and consumption values declined significantly while approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an effective biocide and alternative to Bt toxin.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Effects of gender and temperature on oxidative stress enzymes in Nile tilapia Oreochromis niloticus exposed to paraquat

Many classes of environmental pollutants can enhance the intracellular formation of reactive oxygen species, which can conduce to the damage of macromolecules and changes in oxidant defences levels in fish. In the present study it was analysed the hepatic levels of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione S-transferase (GST) in males and females of Nile tilapia Oreochromis niloticus exposed to paraquat (PQ), at 17 and 27 °C. Tilapia were exposed to a sublethal concentration of PQ (0.5 mg L⁻¹) during 45 days. Condition factor and hepatosomatic index of males and females exposed to PQ were significantly higher when compared with the control group, except in females at 17 °C. SOD and GST activities were higher in males and females exposed to PQ than in the control group at 17 and 27 °C. The levels of both enzyme activities revealed that they are sex-dependent with males exposed to PQ showing higher SOD activity (5.05 ± 0.13 and 4.84 ± 0.23 U/g protein, respectively at 17 and 27 °C) than females (4.21 ± 0.07 and 3.87 ± 0.27 U/g protein, respectively at the same temperature). Similar results were observed in GST activity. A GR activity significantly higher (9.09 ± 0.44 and 7.97 ± 1.08 U/g protein at 17 and 27 °C, respectively) was observed in PQ-exposed females, but not in exposed males. Fish exposed to PQ showed higher values of SOD and GST activities than the control group at both temperatures. These results are gender-dependent, while GR activity was higher only in PQ-exposed females. No significant differences were found for SOD, GST and GR activities between fish exposed to 17 and 27 °C, although males and females showed higher values at 17 °C. In short, this work advanced new knowledge on influence of gender in same biochemical parameters in tilapia exposed to PQ and demonstrated that their effects could be observed at different temperatures.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.     

Plumbagin as a new natural herbicide candidate for Sicyon angulatus control agent with the target 8-amino-7-oxononanoate synthase

A natural compound plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was isolated from the leaves of Plumbago auriculata and found to inhibit the enzyme, 8-amino-7-oxononanoate synthase (AONS, also known as 7-keto-8-aminopelargonate synthase, KAPAS) an IC₅₀ of 2.1 μM in vitro. Biotin supplement significantly rescued the plant injury caused by the plumbagin treatment, and this result confirmed the target site, AONS. Foliar application of 1000 ∼ 2000 μg/mL plumbagin in a greenhouse condition showed lethal activity against eight species of weeds, containing three grass species of Sorghum bicolor, Echinochloa crus-galli, Digitaria sanguinalis and five broad leaf species of Solanum nigrum, Aeschynomene indica, Abutilon avicennae, Xanthium strumarium, Calystegia japonica. Field trial of foliar application with plumbagin 2000 μg/mL have successfully controlled 10 ∼ 15 leaf-stages and 2 ∼ 3 m vine lengths of Sicyos angulatus at the natural habitats around riparian zone in the Nam-Han River in Korea. Visual symptom of desiccation might be induced by the physiological cellular leakage which was significantly dose dependent on the plumbagin treatment regardless of light.     

Activation of paraquat in the earthworm Allolobophora chlorotica is mediated by NAD(P)H-cytochrome c reductase activities

This study reports that earthworms, Allolobophora chlorotica, are capable of biotransforming paraquat, a toxic herbicide, resulting in the formation of reactive oxygen species (ROS). We found that in earthworms the reduction of paraquat is mediated by NADPH- and NADH-cytochrome c reductase activities. The formation of superoxide anion (O₂⁻) from the incubation of paraquat with the earthworm extracts was demonstrated by using both Cypridina luciferin analog (CLA) chemiluminescence and the SOD-inhibitable cytochrome c reduction reaction. In addition, in vivo exposure of earthworms to paraquat in solution (24 and 48 h) was performed to investigate whether or not the herbicide affects the levels of the NAD(P)H-cytochrome c reductase activities. Although in vitro NADPH-cytochrome c reductase reduces paraquat more easily than the NADH-dependent activity, after the in vivo exposure an increase of NADH-cytochrome c reductase activity(s) by 12% compared to control values was observed, whereas NADPH-cytochrome c reductase activity was not affected. Xanthine oxidase (XO) is an enzyme implicated in paraquat toxicity, however, no XO was detected in earthworm extracts nor hypoxanthine was a source of electrons for the herbicide reduction. For comparative reasons menadione, a redox cycling quinone, was also incubated with the earthworm extracts. It was found that the incubation of menadione with earthworm extracts formed about two times more (O₂⁻) than with paraquat. It is concluded that the exposure of paraquat to earthworms could elicit radical formation and consequently toxic effects via oxidative stress-mediated mechanisms. The reduction of paraquat by the reductases leads to the formation of paraquat radical, which reacts with molecular oxygen, accounting for the formation of superoxide anion. Further studies are required to conclude that the observed increase of NADH-cytochrome c reductase activity(s) should be used as a biomarker for paraquat exposure in earthworms.     

Comparison of kinetic properties of a hydrophilic form of acetylcholinesterase purified from strains susceptible and resistant to carbamate and organophosphorus insecticides of green rice leafhopper (Nephotettix cincticeps Uhler)

A hydrophilic form of acetylcholinesterase (AChE) was purified from N-methyl carbamate susceptible (SA) and highly N-methyl carbamate-resistant (N3D) strains of the green rice leafhopper (GRLH), Nephotettix cincticeps Uhler. Both of purified AChE from SA and N3D strains displayed the highest activities toward acetylthiocholine (ATCh) at pH 8.5. In the SA strain, the optimum concentrations for ATCh, propionylthiocholine (PTCh), and butyrylthiocholine (BTCh) were about 1 × 10⁻³, 2.5 × 10⁻³, and 1 × 10⁻³ M, respectively. However, in the N3D strain, substrate inhibition was not identified for ATCh, PTCh, and BTCh to 1 × 10⁻² M. The K_m value in the SA strain was 51.1, 39.1, and 41.6 μM and that in the N3D strain was 91.8, 88.1, and 85.2 μM for ATCh, PTCh, and BTCh, respectively. The K_m value in the N3D strain indicated about 1.80-, 2.25-, and 2.05-fold lower affinity than that of the SA strain for ATCh, PTCh, and BTCh, respectively. The V_max value in the SA strain was 70.2, 30.5, and 4.6 U/mg protein and that in the N3D strain was 123.0, 27.0, and 14.5 U/mg protein for ATCh, PTCh, and BTCh, respectively. The V_max value in the N3D strain was 1.75- and 3.15-fold higher for ATCh and BTCh than that in the N3D strain. However, it was 1.13-fold lower for PTCh. The increased activity of AChE in the N3D strain is due to the qualitatively modified enzyme with a higher catalytic efficiency. The bimolecular rate constant (k_i) for propoxur was 27.1 × 10⁴ and 0.51 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain and that for monocrotophos was 0.031 × 10⁴ and 2.0 × 10⁴ M⁻¹ min⁻¹ in the SA and N3D strain. AChE from the N3D strain was 53-fold less sensitive than SA strain to inhibition by propoxur. In contrast, AChE from the N3D strain was 65-fold more sensitive to inhibition by monocrotophos than AChE from the SA strain. This indicated negatively correlated cross-insensitivity of AChE to propoxur and monocrotophos.     

Methoxyfenozide and pyriproxifen alter the cellular immune reactions of Eurygaster integriceps Puton (Hemiptera: Scutelleridae) against Beauveria bassiana

Effects of the two insect growth regulators (IGRs) methoxyfenozide, 20-hydroxyecdysone (20E) agonist, and pyriproxifen, Juvenile hormone (JH) agonist, were examined on the cellular immune responses of the Sunn pest, Eurygaster integriceps versus the entomopathogenic fungus Beauveria bassiana. The simultaneous treatment with the IGRs and the fungal spores altered haemocyte count (total and differentiate), nodulation response and phenoloxidase (PO) activity in a dose- and time-dependent manner. It was observed that different concentrations of methoxyfenozide increased total and differentiate haemocyte numbers as well as B. bassiana-induced nodulation response. In contrast with the JH agonist, pyriproxifen significantly decreased total and differentiate haemocyte numbers and inhibited nodule formation in E. integriceps adults. The 20E agonist displayed major effects when injected at the doses 2.79 and 5.59 μg/mg adult. In contrast, injecting adults by pyriproxifen significantly impaired their ability to raise an efficacious response against the fungal spores. The ability of the two IGR analogues to interfere with activity of the PO system in haemolymph of E. integriceps adults was also investigated 6 h after injection by fungal spores. Methoxyfenozide had an excitatory effect on PO activity when the 5.59 μg/mg concentration was used against adults. Conversely, pyriproxifen had an inhibitory effect on PO activity when used at 1.49 μg/mg adult concentration. These findings demonstrate that pyriproxifen may interfere with cell-mediated immunity of E. integriceps. So, pyriproxifen could be a good candidate for the integrated control of the Sunn pest.     

Toxicity and biochemical action of amicarthiazol on citrus canker pathogen, Xanthomonas citri ex Hasse

Inhibitory effects of amicarthiazol, in vitro, against the growth of seven plant bacterial pathogens and its biochemical actions on Xanthomonas citri were investigated. The growth of Erwinia carotovora, Ralstonia solanacearum, and Pseudomonas syringe pv. syringae was not sensitive to amicarthiazol whilst Xanthomonads were all strongly inhibited in NB liquid medium; EC₅₀ of X. citri was 2.63 μg ml⁻¹ and the effectiveness of amicarthiazol depended on the timing of application. The inhibitions on the secretion of gross EPS and gross EPR as well as the activities of amylase, cellulase, and pectase of X. citri were increased with the concentrations of amicarthiazol, whereas the conductivities of the extracellular product suspensions and the protease activity were somewhat accelerated by the concentration less than 10 or 20 μg ml⁻¹ and inhibited only at higher concentration. EPS which was purified from X. citri showed obvious antagonistic effects on the inhibition of amicarthiazol, but not on the inhibition of Cu(OH)₂. The endogenous respirations and the activity of succinic dehydrogenase of X. citri were also inhibited and the inhibitions were increased with the toxic concentrations. Furthermore, the potential induced physiological adaptability of X. citri to amicarthiazol and the comparison with Cu(OH)₂ and carboxin were also discussed.     

Structure-activity relationships of pheromonostasis induced by ACCase-inhibitor herbicides in the moth Helicoverpa armigera

Many moth sex pheromone blends are derived from fatty acids and their production is regulated by a Pheromone Biosynthesis Activating Neuropeptide (PBAN). In prior work we showed that the herbicide Diclofop-acid, an acetyl-coenzyme A carboxylase (ACCase) inhibitor, inhibits PBAN-induced sex pheromone production in vitro. In this work we extend our study showing that several other herbicides, belonging to the 2-aryloxyphenoxypropionate (‘FOP’) and the cyclohexandione-oxime (‘DIM’) families significantly inhibit pheromone production by adult females whilst survival is unaffected by treatment. Enzyme activity in vitro and kinetic analysis revealed a K_m of 0.35 μM with K_i values of 0.1 and 0.28 μM due to Tralkoxydim and Diclofop inhibition, respectively. Inhibitory activity on PBAN-induced pheromone production by all herbicides tested revealed a potency order: Tralkoxydim > Clodinafop > Cycloxidim > Haloxyfop > Diclofop > Fenoxaprop > Fluazifop > Quizalofop, Quizalofop being inactive. Differences in inhibition efficiencies may be attributed to different binding sites on the enzyme or to the polarity and solubility of these compounds.     

Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.     

Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L.

The molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of Solanum siniacum and Artemisia judaica L. plants against Biomphalaria alexandrina snails was carried out. The tests revealed plant’s ethanol extract was more toxic to the snails than the other tested extracts. Therefore, it was tested against snails’ fecundity (M_x), reproduction rate (R₀) and their infection with Schistosoma mansoni miracidia. In addition, biochemical parameters and the activities of some enzymes in tissues of snails treated with the two tested plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of B. alexandrina snails to plant’s ethanol extract led to a significant reduction in their survival and snails’ fecundity, reproduction rate. In addition, it caused a considerable reduction in the infectivity of S. mansoni miracidia to the snails. Also, it caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results revealed that the glucose concentration in hemolymph and Lactate level in soft tissues of treated snails were increased (P < 0.001) while glycogen, total protein, the lipid content and the pyruvate level in snail’s tissues decreased (P < 0.001). The activities of lactic dehydrogenase (LDH), pyruvatekinase (PK) and cytochrome oxidase (CY) enzymes in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with the two tested plants while protease (PR) activity increased (P < 0.001). It is concluded that the application of LC₂₅ of ethanol extract of S. siniacum and A. judaica L. may be helpful in snail control as it interferes with the snails’ biology and physiology.     

Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L.

The efficacy of the essential oil and various organic extracts from flowers of Cestrum nocturnum L. was evaluated for controlling the growth of some important phytopathogenic fungi. The oil (1000 ppm) and the organic extracts (1500 μg/disc) revealed antifungal effects against Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum in the growth inhibition range of 59.2-80.6% and 46.6-78.9%, respectively, and their MIC values were ranged from 62.5 to 500 and 125 to 1000 μg/mL. The essential oil had a remarkable effect on spore germination of all the plant pathogens with concentration and time-dependent kinetic inhibition of P. capsici. Further, the oil displayed remarkable in vivo antifungal effect up to 82.4-100% disease suppression efficacy on greenhouse-grown pepper plants. The results obtained from this study may contribute to the development of new antifungal agents to protect the crops from fungal diseases.     

Effects of insecticides on plant-growth-promoting activities of phosphate solubilizing rhizobacterium Klebsiella sp. strain PS19

In this study, four technical grade insecticides, fipronil, pyriproxyfen, imidacloprid and thiamethoxam were applied at the recommended and the higher doses to investigate their effects on plant growth-promoting activities of phosphate-solubilizing Klebsiella sp. strain PS19, isolated from mustard rhizosphere. All tested insecticides displayed a concentration-dependent inhibition in plant growth promoting traits, like, inorganic phosphate solubilization, biosynthesis of phytohormones and siderophores, of rhizobacterial strain PS19. For example, the phosphate-solubilizing activity of Klebsiella sp. PS 19 was reduced maximally by 95%, at 3900 μg l⁻¹ pyriproxyfen over control. At the recommended rate, the magnitude of toxicity of insecticides to plant growth promoting traits was less severe compared to the higher doses. The sequence of insecticide-toxicity expressed as percent decrease, determined at highest dose rate of each insecticide, over control was: pyriproxyfen (95) = imidacloprid (95) > thiamethoxam (94) > fipronil (85), for phosphate-solubilizing activity while for salicylic acid (SA) it was: thiamethoxam > pyriproxyfen = imidacloprid > fipronil. The impact of the highest dose rate of insecticides on 2,3-dihydroxybenzoic acid (DHBA) was almost equal to those observed for SA. Thiamethoxam decreased the indole acetic acid (IAA) synthesis maximally by 86% whereas fipronil had least toxicity and reduced it by 67% relative to the control. Among the experimental insecticides, pyriproxyfen at 3900 μg l⁻¹ in general, had the greatest toxic effects for plant growth promoting activities of the test strain. The study inferred that insecticides affect the plant beneficial activities of rhizobacteria adversely. These findings are likely to add a new insight into the pest management practices.     

Direct measurement of paraquat in leaf protoplasts indicates vacuolar paraquat sequestration as a resistance mechanism in Lolium rigidum

Sequestration of paraquat away from its target site in the chloroplast has been proposed as a mechanism of paraquat resistance. However, no consensus has been reached as to where paraquat is sequestered. This study quantifies paraquat in leaf protoplasts of paraquat resistant (R) and susceptible (S) Lolium rigidum. Intact protoplasts were prepared from plants treated with commercial dose of paraquat for 2 h. Paraquat absorbed by the leaf protoplasts was determined by light absorption of reduced paraquat following concentration and purification using a cation-exchange resin. Leaf protoplasts from treated paraquat resistant plants contained 2- to 3-fold more paraquat than leaf protoplasts isolated from susceptible plants. Since paraquat is not metabolised in L. rigidum and paraquat readily enters chloroplasts of both R and S plants, this greater amount of paraquat in leaf protoplasts of R plant must be kept away from the target site (chloroplast). This result indicates that paraquat resistance in L. rigidum is associated with a cytoplasmic mechanism, most likely a greater rate of vacuolar sequestration.     

Effect of fenvalerate on oxidative stress biomarkers in the brackish water prawn Penaeus monodon

The toxicity of fenvalerate to the prawn Penaeus monodon was evaluated using biomarkers of stress. In a preliminary bioassay test, P. monodon was exposed to a series of fenvalerate concentrations, which showed 4, 6.5 and 8.5 μg L⁻¹ to be sublethal, median lethal and lethal, respectively. Sublethal effect of fenvalerate was further evaluated in hepatopancreas, muscle and gills of prawns with reference to oxidative stress biomarkers. Significant induction of lipid peroxidation and glutathione-S-transferase activity was found in hepatopancreas, muscle and gills of prawns exposed to fenvalerate when compared to control (P < 0.001, P < 0.05 and P < 0.05). On the contrary, the activities of Superoxide dismutase, catalase, glutathione peroxidase, vitamin C, vitamin E and glutathione were found to be reduced in the experimental group of prawns when compared to control. The results suggest that the animals were under oxidative stress when exposed to sublethal concentration of fenvalerate.     

Effect of dihydrodillapiole on pyrethroid resistance associated esterase inhibition in an Indian population of Spodoptera litura (Fabricius)

Resistance in Spodoptera litura (Fabricius) has been attributed to enhanced detoxification of insecticides by increased levels of esterases, oxidases and/or glutathione S-transferases. Enzyme inhibiting insecticide synergists can be employed to counter increased levels of such enzymes in S. litura. Dihydrodillapiole induced synergism of pyrethroid toxicity was examined in the laboratory-reared third instar larval population of S. litura collected in Delhi (susceptible), and Guntur (resistant) region of Andhra Pradesh, India. The Guntur population was found to be 7.04 and 10.19 times resistant to cypermethrin and lambdacyhalothrin, respectively. The activity of cypermethrin, lambdacyhalothrin and profenophos against susceptible and resistance populations of S. litura, was gradually increased when used along with a plant-derived insecticide synergist dihydrodillapiole. The α-naphthyl acetate hydrolysable esterase activity in Delhi population was less as compared to the Guntur population. Resistance associated esterases in Delhi population were inhibited by pre-treatment with dihydrodillapiole. The esterase level in insect was instantly reduced initially, sustained for about 3 h and equilibrated at 4 h post treatment. The esterase activity of Guntur population was increased to 1.28 μmoles/mg/min at 2 h post treatment and subsequently reduced to lower than 0.70 μmoles at 4-12 h post treatment. The variation in esterase activity is suggestive of its homeostatic regulation in test populations. Dihydrodillapiole thus caused significant reduction of resistance in S. litura to cypermethrin, lambda cyhalothrin and profenophos.     

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures,followed by modeling of ecdysteroid-EcR interactions and normal mode analysis

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC₅₀ = 3.3 μM) and Bm5 cells (EC₅₀ = 5.3 μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC₅₀ = 0.71 μM and 0.00089 μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC₅₀ of 0.039 μM; in Bm5 cells this effect only became visible at much higher concentrations (IC₅₀ = 18 μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.