Ellen De Geyter

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells

This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of Sclerotinia sclerotiorum, referred to as S. sclerotiorum agglutinin or SSA. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC50) of 66 (49-88) μg/ml. In an attempt to unravel the mode of action of SSA the binding and interaction of the lectin with insect tissues and cells were investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 μg/ml SSA resulted in a total loss of cell viability, the median cell toxicity value (EC50) being 4.0 (2.4-6.7) μg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell surface. Prior incubation of the cells with saponin to achieve a higher cell membrane permeation resulted in an increased internalization of SSA in the insect midgut cells, but no increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was significantly reduced when SSA was incubated with GalNAc and asialomucin prior to treatment of the cells, the data of this project provide strong evidence that SSA binds with specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction cascade leading to death of the midgut epithelial cells, which in turn results in insect mortality. The potential use of SSA in insect control is discussed.

Triterpene saponins of Quillaja saponaria show strong aphicidal and deterrent activity against the pea aphid Acyrthosiphon pisum

BACKGROUND Saponins are a class of secondary plant metabolites consisting of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin) that often possess insecticidal activities. Four saponins were selected: two triterpene saponins, Q. saponaria saponins and aescin, and two steroidal saponins, digitonin and diosgenin. Their effects were investigated on an important pest species and a model piercing-sucking insect, the pea aphid Acyrthosiphon pisum. The triterpene Q. saponaria saponins bark saponin received special attention because of its high activity. Aphids were challenged by oral and contact exposure to demonstrate aphicidal activities, and in choice experiments to support use as a natural deterrent. RESULTS When aphids were exposed to supplemented artificial diet for 3 days, a strong aphicidal activity was recorded for three of the four saponins, with an LC50 of 0.55 mg mL(-1) for Q. saponaria saponins, 0.62 mg mL(-1) for aescin and 0.45 mg mL(-1) for digitonin. The LT50 values ranged between 1 and 4 days, depending on the dose. For diosgenin, only low toxicity (14%) was scored for concentrations up to 5 mg mL(-1). In choice experiments with treated diet, a deterrence index of 0.97 was scored for Q. saponaria saponins at 1 mg mL(-1). In contrast, direct contact showed no repellent effect. Spraying of faba bean plants with Q. saponaria saponins resulted in an LC50 of 8.2 mg mL(-1). Finally, histological analysis in aphids fed with Q. saponaria saponins demonstrated strong aberrations of the aphid gut epithelium, and exposure of midgut CF-203 cell lines to Q. saponaria saponins in vitro confirmed the cytotoxic effect. CONCLUSIONS The present insect experiments provide strong evidence that saponins, as tested here with triterpene Q. saponaria saponins, can be useful as natural aphicides and deterrents. Furthermore, the insect midgut epithelium is suggested to be a primary target of saponin activity.

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays

BACKGROUND Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species-specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS When collections of non-steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100-fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.

Saponins do not affect the ecdysteroid receptor complex but cause membrane permeation in insect culture cell lines.

This project studied the effects of four saponins with a triterpenoid (Quillajasaponaria saponin and aescin) or steroid structure (digitonin and diosgenin which is the deglycosylated form of dioscin) on insect cells, namely Schneider S2 cells of Drosophila melanogaster (Diptera). A series of different experiments were performed to investigate potential mechanisms of action by saponins with regard to ecdysteroid receptor (EcR) responsiveness, cell viability, cell membrane permeation, and induction of apoptosis with DNA fragmentation and caspase-3 like activity. Major results were that (1) exposure of S2 cells containing an EcR-based reporter construct to a concentration series of each saponin scored no EcR activation, while (2) a loss of ecdysteroid signaling was observed with median inhibitory concentrations (IC(50)s) of 3-50 μM, and in parallel (3) a concentration-dependent change in loss of cell numbers in an cell viability assay with median effective concentrations (EC(50)s) of 8-699 μM. In continuation, it was of interest that (4) a trypan blue assay with Q. saponaria saponin confirmed the cell membrane permeation effect leading to cell toxicity with a median lethal concentration (LC(50)) value of 44 μM, and interestingly this effect was very rapid. Another three interesting observations were that (5) exposure to 20E at 500 nM as used in the EcR-based report assay induced caspase-3 like activities which may help to explain the discrepancies between loss of EcR-responsiveness and cell viability, (6) low concentrations of saponins induced DNA fragmentation and caspase-3 like activities, confirming their potential to induce apoptosis, and (7) the saponin effects were counteracted with addition of cholesterol to the culture medium. In general the data obtained provide evidence that the anti-ecdysteroid action by saponins is not based on a true antagonistic interaction with EcR signaling, but can be explained by a cytotoxic action due to permeation of the insect cell membrane.

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=3.3μM) and Bm5 cells (EC50=5.3μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71μM and 0.00089μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039μM; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18μM). 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.

Saponins show high entomotoxicity by cell membrane permeation in Lepidoptera

BACKGROUND In this study, the effects of three saponins and one sapogenin with a triterpenoid or steroid structure in two lepidopteran insect cell lines, ovarian Bm5 and midgut CF-203 cells, were analysed with regard to cell viability, cell membrane permeation, EcR responsiveness and DNA fragmentation. In addition, the entomotoxic action of Q. saponaria saponin with primary midgut cell cultures and larval stages of the cotton leafworm Spodoptera littoralis was tested. RESULTS Both lepidopteran cell lines show a high sensitivity to all four sapo(ge)nins, with a concentration-dependent viability loss and EC₅₀ values of 25-100 µM in MTT bioassays. A trypan blue assay with Q. saponaria saponin confirmed rapid cell membrane permeation to be a cause of cytotoxicity. Saponins caused no EcR activation in Bm5 cells, but a loss of ecdysteroid signalling was observed with IC₅₀ values of 5-10 µM. Lower saponin concentrations induced DNA fragmentation, confirming their potential to induce apoptosis. Finally, Q. saponaria saponin caused cytotoxicity in primary midgut cell cultures of S. littoralis (EC(50) = 4.7 µM) and killed 70-84% of S. littoralis larvae at pupation at 30-70 mg g(-1) , while lower concentrations retarded larval weight gain and development. CONCLUSIONS The data obtained provide evidence that saponins exert a strong activity on lepidopteran cells, presumably based on a cytotoxic action due to permeation of the cell membrane. Primary midgut cell cultures and larvae of S. littoralis showed high sensitivity to Q. saponaria saponin, indicating the insect midgut as a primary target for entomotoxicity and the potential use of saponins in the control of pest Lepidoptera.

Insecticidal Activity of Saponins

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in citrus. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x×4x hybridisations and in vitro culture of isolated nucellus. The origin of the plants obtained as a consequence of these two processes is still unclear. In this work, we systematically analyzed the genetic structure (ploidy and allelic constitution at SSR locus) of plants obtained from polyembryonic seeds arising from 2x×4x sexual hybridisations or regenerated from nucellus culture in vitro of different non-apomictic citrus genotypes. Histological studies were also conduced to try to identify the initiation process of polyembryony in nonapomictic genotypes. We demonstrate that all plants obtained from the same undeveloped seed in 2x×4x hybridisations resulted from fission of the original zygotic embryo. Also, the plants obtained from in vitro culture of nucellus were recovered by somatic embryogenesis from cells having the same genotype as the zygotic embryos of the same seed. It appears that in non-apomictic citrus, proembryos or embryogenic cells are formed by fission of the original zygotic embryo and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as result of two different mechanisms that prevent the dominance of the initial zygotic embryo. (Texte integral)