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protein content was determined by the Kjeldahl method. The phenol sulfuric acid method was used to estimate total carbohydrate content using glucose as a standard. Total lipid content was measured using the sulphophosphovanillin method. All the assays were performed in duplicate and repeated thrice. H. armigera enzyme activity assays Whole larval tissues were homogenized in 300 mL of 0.02 M sodium-phosphate buffer containing 10 mM NaCl for 2 h at 4uC. The homogenate was then centrifuged at 13,0006g for 30 min at 4uC. The supernatant was collected, stored at 220uC and used as crude enzyme source. Total protease activity from H. armigera larvae fed on O. kilimandscharicum plants was assayed using azocasein as substrate. Trypsin activity assays were performed as described by Tamhane et al.. One protease unit was defined as the amount of enzyme in the assay that causes an increase in absorbance by one optical density under the given assay conditions. Amylase Plant maintenance O. kilimandscharicum and tomato plants were grown in the greenhouse. The conditions in the greenhouse were as follows: temperature, 28 to 30uC; humidity, 35 to 40%; light conditions, 16 h light, 8 h dark. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19664521 Insecticidal Metabolites from O. kilimandscharicum activity from the gut of H. armigera larvae was analyzed by the dinitrosalycylic acid method, as described by Kotkar et al.. One amylase unit was defined as the amount of enzyme required to release 1 mM maltose/minute at 37uC under the given assay conditions. Lipase activity from gut homogenates was estimated using the p164 nitrophenyl palmitate assay. One unit of lipase activity was defined as the amount of enzyme that causes an increase of one optical density under the given assay conditions. Nitrogen was used as the carrier gas at a flow rate of 1 mL/min. The column temperature was raised from 70uC to 110uC at 2uC min21, then raised to 180uC at 3uCmin21 and finally to a temperature of 220uC with a 10uC min21 rise; here it was held for 2 min. Injector and detector temperatures were 230uC and 250uC, respectively. GC-MS was performed on a HP 5975C mass selective detector interfaced with a HP 7890A gas chromatograph. GC-MS analyses were performed under similar conditions using an HP-5 MS capillary column with helium as the carrier gas. Compounds were identified by comparing the retention time and mass fragmentation pattern of the Triptolide standards of major constituents and also by comparing acquired mass spectra and retention indices with NIST/NBS and the Wiley mass spectral library. average larval body mass were recorded every alternate day up to pupation. Pupal deformities were also recorded. Statistical analysis Significant differences between diet treatments were determined using one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparison and mentioned in respective figure legends. Unpaired T test was used to compare data from two treatments i. e. tomato and O. kilimandscharicum in respective analysis and to compare metabolic changes in local and systemic tissue, and detailed information provided in respective figure legends. One way ANOVA and Unpaired t-test data was considered to be significantly different within the treatments if the F-value obtained was higher than the critical F-value at p,0.001, p,0.01, p,0.05. Small letters are used to indicate statistically different groups of treatments. NS represents non-significant difference within the treatments and/or in the respective day. Results and Discussion

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Author: M2 ion channel