K. Sreeramulu

P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: purification, characterization and effect of various insecticides on its transport function.

Helicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30-40 degrees C, respectively. Kinetic studies indicated that this enzyme had a K(m) value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest.

Differential elicitation of proteases and protease inhibitors in two different genotypes of chickpea (Cicer arietinum) by salicylic acid and spermine.

Elicitation of proteases and protease inhibitors (PIs) by salicylic acid (SA) and spermine (Spm) was investigated in roots and shoots of two different genotypes of chickpea cultivars ICCV10 and L550, which were resistant and susceptible to wilt disease, respectively. SA and Spm were found to suppress the elicitation of proteases in the resistant cv, whereas they induce it in susceptible cv. Elicitation of new trypsin and chymotrypsin inhibitors was observed in the roots and shoots of resistant cv treated with SA and Spm. However, no such elicitation was observed in susceptible cv. These results show for the first time that SA and Spm could elicit synthesis of new PIs capable of inhibiting the proteases of insect Helicoverpa armigera and Fusarium oxysporum, wilt causing pathogen. Antifungal property of root extract of resistant cv increased following treatment of seedling with SA and Spm compared with susceptible cv.

Interaction of plant cell signaling molecules, salicylic acid and jasmonic acid, with the mitochondria of Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera is a polyphagous pest in Asia, Africa, and the Mediterranean Europe. Salicylic acid (SA) and jasmonic acid (JA) are the cell signaling molecules produced in response to insect attack in plants. The effect of these signaling molecules was investigated on the oxidative phosphorylation and oxidative stress of H. armigera. SA significantly inhibited the state III and state IV respiration, respiratory control index (RCI), respiratory complexes I and II, induced mitochondrial swelling, and cytochrome c release in vitro. Under in vivo conditions, SA induced state IV respiration as well as oxidative stress in time- and dose-dependent manner, and also inhibited the larval growth. In contrast, JA did not affect the mitochondrial respiration and oxidative stress. SA affected the growth and development of H. armigera, in addition to its function as signaling molecules involved in both local defense reactions at feeding sites and the induction of systemic acquired resistance in plants.

Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner)

Helicoverpa armigera is one of the most important pests worldwide. Transgenic crops with toxin genes from Bacillus thuringiensis (Bt) have been deployed on a large scale to control this pest. The insecticidal activity of Bt is probably influenced by the insect midgut microbes, which vary across crop hosts and locations. Therefore, we examined the role of gut microbes in pathogenicity of Bt toxins in the H. armigera. Antibiotic cocktail was used for the complete elimination of the H. armigera gut microbes. Activated Cry1Ac, Bt formulation, and transgenic cotton resulted in larval weight loss and increase in mortality, but pretreatment of larvae with antibiotic cocktail significantly decreased larval mortality and increased the larval weight gain. Activated Cry1Ac and Bt formulation inhibited the activity of proteases in midgut of H. armigera larvae but showed no such effect in the larvae pretreated with antibiotic cocktail. Five protease bands in activated Cry1Ac and two in Bt formulation-treated larvae were inhibited but no such effect in the larvae pretreated with antibiotic cocktail. Cry1Ac protein was detected in Bt/Cry1Ac protoxin-fed larval gut extract in the absence of antibiotic cocktail, but fewer in larvae pretreated with antibiotic cocktail. The activity of antioxidant enzymes and aminopeptidases increased in larvae fed on Bt toxin, but there was no significant increase in antioxidant enzymes in larvae reared on toxin protein in combination with antibiotic cocktail. The results suggest that gut microbes exercise a significant influence on the toxicity of Cry1Ac and Bt formulation in H. armigera larvae. The implications of these results have been discussed in relation to development of insect resistance to Bt transgenic crops deployed for pest management.

MODULATION OF P‐GLYCOPROTEIN ATPASE OF HELICOVERPA ARMIGERA BY CHOLESTEROL: EFFECTS ON ATPASE ACTIVITY AND INTERACTION OF INSECTICIDES

Purified P-glycoprotein ATPase from Helicoverpa armigera (Ha-Pgp), reconstituted in proteoliposomes composed of phospholipids and cholesterol, shows higher ATPase activity in the presence of cholesterol than in its absence. The Ha-Pgp ATPase activity was increased 30-40% with cholesterol. The KM for ATP was found to be 1 and 0.8 mM in the absence and presence of cholesterol, respectively. The insecticide-stimulated Ha-Pgp ATPase activity was increased by 10-20% for all the insecticides in the reconstituted proteoliposomes containing cholesterol compared to those with no cholesterol. The effects of cholesterol on KM and Vmax values of insecticide-stimulated Ha-Pgp ATPase activity were unrelated to the size of the insecticide. Ha-Pgp tryptophan fluorescence displayed a red shift of 3 and 8 nm in emission spectra upon binding of insecticides. Cholesterol enhances the interaction of insecticides with Ha-Pgp. Kd values of different insecticides for binding to Ha-Pgp were found to be lower in the presence of cholesterol in the proteoliposomes compared to its absence. Results suggest that cholesterol plays a role in the recognition and interaction of insecticides by modulating Ha-Pgp ATPase and may be involved in efflux of insecticides from cells by the transporter.

Purification and characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera

Phenoloxidases are oxidative enzymes, which play an important role in both cell mediated and humoral immunity. Purification and biochemical characterization of prophenoloxidase from cotton bollworm, Helicoverpa armigera (Hübner) were carried out to study its biochemical properties. Prophenoloxidase consists of a single polypeptide chain with a relative molecular weight of 85 kDa as determined by SDS–PAGE, MALDI–TOF MS and LC–ESI MS. After the final step, the enzyme showed 71.7 fold of purification with a recovery of 49.2%. Purified prophenoloxidase showed high specific activity and homology with phenoloxidase subunit‐1 of Bombyx mori and the conserved regions of copper binding (B) site of phenoloxidase. Purified prophenoloxidase has pH optima of 6.8 and has high catalytic efficiency towards the dopamine as a substrate in comparison to catechol and L‐Dopa. The PO activity was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and kojic acid.

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R1-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H2O2 concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H2O2 in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.

Characterization of dihydrolipoamide dehydrogenase from the mitochondria of Helicoverpa armigera, a pest resistant to insecticides

Dihydrolipoamide dehydrogenase (DHLDH) was isolated from the mitochondria of Helicoverpa armigera, a destructive pest which has developed resistance to commonly used insecticides. The flavoenzyme was purified 17.98‐fold to homogeneity with an overall yield of 10.53% by employing ammonium sulfate precipitation, hydroxylapatite chromatography and CM‐Sephadex chromatography. The purified enzyme exhibited the specific activity of 18.7 U/mg and was characterized as a dimer with a subunit mass of 66 kDa. The enzyme showed specificity for nicotinamide adenine dinucleotide – hydrogen (NADH) and lipoamide, as substrates, with Michaelis‐Menten constants (Km) of 0.083 mmol/L and 0.4 mmol/L, respectively. The reduction reaction of lipoamide by the enzyme could be explained by ping‐pong mechanism. The spectra of DHLDH showed the maximum absorbance at 420 nm, 455 nm and 475 nm. The enzyme activity was strongly inhibited by mercurial and arsenical compounds. The N‐terminal sequence of Ha‐DHLDH showed homology with those of mammalian and arthropod DHLDH. Since H. armigera has developed high levels of resistance to commonly used insecticides, biochemical properties of the metabolic enzymes such as DHLDH, could be helpful to develop insecticidal molecules for the control of H. armigera, with a different mode of action.

Inhibition of Catalase Isozymes of Helicoverpa armigera(Lepidoptera: Noctuidae) by Salicylic acid.

The cotton bollworm, Helicoverpa armigera is a polyphagous pest and causes oxidative damage to field crops. Salicylic acid is the cell signaling molecule produced in response to insect attack on plants. Catalase plays a major role in the protection of tissues from the toxic effects of H2O2 and partially reduced oxygen species. Invitroand Invivo effect of salicylic acid was studied on the activities of catalases of Helicoverpa armigera, and tissue distribution of catalase isoforms was also studied. Eight catalase isoforms were found in the gut and whole body preparations whereas single isoforms in the hemolymph. Salicylic acid inhibited over 90 % of catalase activity at 2 mM concentrations in the gel activity staining as well as spectrophotometrically, but catalase I in all the preparations are stable and insensitive to salicylic acid.

Evaluation of flubendiamide-induced mitochondrial dysfunction and metabolic changes in Helicoverpa armigera (Hubner)

Phthalic acid diamide insecticides are the most effective insecticides used against most of the lepidopteran pests including Helicoverpa armigera, a polyphagous pest posing threat to several crops worldwide. The present studies were undertaken to understand different target sites and their interaction with insect ryanodine receptors (RyR). Bioassays indicated that flubendiamide inhibited the larval growth in dose-dependent manner with LD50 value of 0.72 μM, and at 0.8 μM larval growth decreased by about 88%. Flubendiamide accelerated the Ca2+ -ATPase activity in dose-dependent trend, and at 0.8 μM, the activity was increased by 77.47%. Flubendiamide impedes mitochondrial function by interfering with complex I and F0 F1 -ATPase activity, and at 0.8 μM the inhibition was found to be about 92% and 50%, respectively. In vitro incubation of larval mitochondria with flubendiamide induced the efflux of cytochrome c, indicating the mitochondrial toxicity of the insecticide. Flubendiamide inhibited lactate dehydrogenase and the accumulation of H2 O2 , thereby preventing the cells from lipid peroxidation compared to control larvae. At 0.8 μM the LDH, H2 O2 content and lipid peroxidation was inhibited by 98.44, 70.81, and 70.81%, respectively. Cytochrome P450, general esterases, AChE, and antioxidant enzymes (catalase and superoxide dismutase) exhibited a dose-dependent increasing trend, whereas alkaline phosphatase and the midgut proteases, except amino peptidase, exhibited dose-dependent inhibition in insecticide-fed larvae. The results suggest that flubendiamide induced the harmful effects on the growth and development of H. armigera larvae by inducing mitochondrial dysfunction and inhibition of midgut proteases, along with its interaction with RyR.