Toxicity and mode of action of steroid and terpenoid secondary plant metabolites against economically important pest insects in agriculture

In the light of the ever-growing problem of insect resistance against the most commonly used groups of insecticides, there is much interest in the development of new alternatives to slow down the trend towards resistance build-up. In order to search for new insect-specific pesticides, we employed an in vitro cell-based reporter bioassay for screening for potential ecdyson receptor (EcR) agonistic and antagonistic activity in natural ecdysteroids. We were able to confirm the validity of the bioassay by identifying one EcR agonistic compound (cyasterone) and one antagonistic (castasterone). Three-dimensional modelling of their interaction with the ecdysone receptor confirmed these findings. However, the EcR activity of before mentioned compounds was low compared to commercial insecticides. Another potentially interesting class of natural molecules are the saponins, a group of secondary plant metabolites consisting of a sugar moiety glycosidically linked to a hydrophobic aglycone. We studied their effects on one dipteran (Schneider S2 cells) and two lepidopteran insect cell lines (ovarian Bm5 cells and midgut CF-203 cells). Major results were that exposure to saponins caused no EcR activation, but did result in a dose-dependent loss of ecdysteroid signalling. In parallel, we saw a similar loss of cell activity in MTT cell viability assays. A trypan blue assay confirmed that Quillaja saponaria saponins caused cell membrane permeation, which lead to the conclusion that the anti-ecdysteroid action by saponins is not based on a direct antagonistic interaction with EcR signalling, but rather on a cytotoxic effect due to permeation of the insect cell membrane. In another series of experiments, we investigated the effects of saponins in vivo on two important insect pest species: the pea aphid Acyrthosiphon pisum (Hemiptera), and the cotton leafworm Spodoptera littoralis (Lepidoptera). When aphids were exposed to supplemented artificial diet for 3 days, a strong aphicidal activity was recorded for three of the four saponins. For caterpillars, third instars of S. littoralis fed with Q. saponaria saponins at a dose of 30-70 mg/g in the diet showed a significant reduction in larval weight gain already after 1 day of treatment, and this negative effect continued during subsequent feeding, leading to 70-84% mortality at pupation. In choice experiments with saponin-treated versus untreated artificial diet, aphids showed a very pronounced preference for the untreated diet, revealing a strong deterrent effect. Similar results were found when saponins were sprayed on bean plant leaves. Looking for the cause behind the insect toxicity, we examined the midgut-specific effects of Q. saponaria saponins. Primary midgut cell cultures of S. littoralis showed concentration-dependent symptoms of cytotoxicity under the confocal fluorescence microscope. Histological analysis using microscopic slides of the A. pisum aphid gut confirmed that the epithelium was severy damaged. These results suggest the insect midgut epithelium to be a primary target of saponin activity. We conclude that saponins (especially Q. saponaria) have a strong and fast-acting effect on the pea aphid A. pisum and the cotton leafworm S. littoralis, most likely due to a combination of deterrent activity and cell membrane permeation leading to destruction of the cells of the insect midgut epithelium, causing the insect to starve and die. These observations provide strong evidence that saponins are natural insecticides and deterrents. For them to be successful in the control of pest insects within Integrated Pest Management, trials under field conditions are needed as well as an evaluation of the possible risks to natural enemies, the environment and the human health

Plasma modification of poly lactic acid solutions to generate high quality electrospun PLA nanofibers

Physical properties of pre-electrospinning polymer solutions play a key role in electrospinning as they strongly determine the morphology of the obtained electrospun nanofibers. In this work, an atmospheric-pressure argon plasma directly submerged in the liquid-phase was used to modify the physical properties of poly lactic acid (PLA) spinning solutions in an effort to improve their electrospinnability. The electrical characteristics of the plasma were investigated by two methods; V-I waveforms and Q-V Lissajous plots while the optical emission characteristics of the plasma were also determined using optical emission spectroscopy (OES). To perform a complete physical characterization of the plasma-modified polymer solutions, measurements of viscosity, surface tension, and electrical conductivity were performed for various PLA concentrations, plasma exposure times, gas flow rates, and applied voltages. Moreover, a fast intensified charge-couple device (ICCD) camera was used to image the bubble dynamics during the plasma treatments. In addition, morphological changes of PLA nanofibers generated from plasma-treated PLA solutions were observed by scanning electron microscopy (SEM). The performed plasma treatments were found to induce significant changes to the main physical properties of the PLA solutions, leading to an enhancement of electrospinnability and an improvement of PLA nanofiber formation

Species-specific differences in follicular antral sizes result from diffusion-based limitations on the thickness of the granulosa cell layer

The size of mature oocytes is similar across mammalian species, yet the size of ovarian follicles increases with species size, with some ovarian follicles reaching diameters more than 1000-fold the size of the enclosed oocyte. Here we show that the different follicular sizes can be explained with diffusion-based limitations on the thickness of the hormone-secreting granulosa layer. By analysing published data on human follicular growth and granulosa cell expansion during follicular maturation we find that the 4-fold increase of the antral follicle diameter is entirely driven by an increase in the follicular fluid volume, while the thickness of the surrounding granulosa layer remains constant at about 45+/-10 mkm. Based on the measured kinetic constants, the model reveals that the observed fall in the gonadotropin concentration from peripheral blood circulation to the follicular antrum is a result of sequestration in the granulosa. The model further shows that as a result of sequestration, an increased granulosa thickness cannot substantially increase estradiol production but rather deprives the oocyte from gonadotropins. Larger animals (with a larger blood volume) require more estradiol as produced by the ovaries to downregulate FSH-secretion in the pituitary. Larger follicle diameters result in larger follicle surface areas for constant granulosa layer thickness. The reported increase in follicular surface area in larger species indeed correlates linearly both with species mass and with the predicted increase in estradiol output. In summary, we propose a structural role for the antrum in that it determines the volume of the granulosa layer and thus the level of estrogen production.Comment: Mol Hum Repr 201