Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery.

Influence of CO2 and Temperature on Metabolism and Development of Helicoverpa armigera (Noctuidae: Lepidoptera).

Climate change will have a major bearing on survival and development of insects as a result of increase in CO2 and temperature. Therefore, we studied the direct effects of CO2 and temperature on larval development and metabolism in cotton bollworm, Helicoverpa armigera (Hübner). The larvae were reared under a range of CO2 (350, 550, and 750 ppm) and temperature (15, 25, 35, and 45°C) regimes on artificial diet. Elevated CO2 negatively affected the larval survival, larval weight, larval period, pupation, and adult emergence, but showed a positive effect on pupal weight, pupal period, and fecundity. Increase in temperature exhibited a negative effect on larval survival, larval period, pupal weights, and pupal period, but a positive effect on larval growth. Pupation and adult emergence were optimum at 25°C. Elevated CO2 and temperature increased food consumption and metabolism of larvae by enhancing the activity of midgut proteases, carbohydrases (amylase and cellulase), and mitochondrial enzymes and therefore may cause more damage to crop production. Elevated CO2 and global warming will affect insect growth and development, which will change the interactions between the insect pests and their crop hosts. Therefore, there is need to gain an understanding of these interactions to develop strategies for mitigating the effects of climate change.

Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera).

Cotton bollworm, Helicoverpa armigera, is one of the most damaging polyphagous pests worldwide, which has developed high levels of resistance to commonly applied insecticides. Mitochondrial P-glycoprotein (Pgp) was detected in the insecticide-resistant strain of H. armigera using C219 antibodies, and its possible role was demonstrated in the efflux of xenobiotic compounds using spectrofluorometer. The TMR accumulated in mitochondria in the absence of ATP, and effluxed out in presence of ATP; the process of efflux was inhibited in the presence of ortho-vandate, an inhibitor of Pgp, in insecticide-resistant larvae of H. armigera. The mitochondria isolated from insecticide-resistant larvae were resistant to insecticide-induced inhibition of oxygen consumption and cytochrome c release. Membrane potential decreased in a dose-dependent manner in the presence of higher concentration of insecticides (>50 µM) in mitochondria of insecticide-resistant larvae. In conclusion, mitochondrial Pgp ATPase detected in the insecticide-resistant larvae influenced the efflux of xenobiotic compounds. Pgp might be involved in protecting the mitochondrial DNA and the components of the electron transport chain from damage due to insecticides, and contributing to the resistance to the deleterious effects of insecticides on the growth of insecticide-resistant H. armigera larvae.