Luc Swevers

Colorado potato beetle (Coleoptera) gut transcriptome analysis: expression of RNA interference-related genes.

In the search for new methods of pest control, the potential of RNA interference (RNAi) is being explored. Because the gut is the first barrier for the uptake of double‐stranded (ds)RNA, pyrosequencing of the gut transcriptome is a powerful tool for obtaining the necessary sequences for specific dsRNA‐mediated pest control. In the present study, a dataset representing the gut transcriptome of the Colorado potato beetle (CPB; Leptinotarsa decemlineata) was generated and analysed for the presence of RNAi‐related genes. Almost all selected genes that were implicated in silencing efficiency at different levels in the RNAi pathway (core machinery, associated intracellular factors, dsRNA uptake, antiviral RNAi, nucleases), which uses different types of small RNA (small interfering RNA, microRNA and piwi‐RNA), were expressed in the CPB gut. Although the database is of lower quality, the majority of the RNAi genes are also found to be present in the gut transcriptome of the tobacco hornworm [TH; Manduca sexta (19 out of 35 genes analysed)]. The high quality of the CPB transcriptome database will lay the foundation for future gene expression and functional studies regarding the gut and RNAi.

Use of RNAi for Control of Insect Crop Pests

RNA interference (RNAi) refers to double-stranded RNA (dsRNA)-mediated gene silencing. Since its discovery, it has developed as a powerful tool in functional genomics, and to date it is widely used in insect genetic research. It is certain that the discovery of RNAi has augmented our understanding of ~20–30 nucleotide non-coding small RNA as critical regulators of gene expression and genome stability. Besides, gene silencing through RNAi has revolutionized the study of gene function, particularly in non-model and non-genome sequenced insect species, which is the case for most agricultural pest insects. Without doubt, it contains great potential for diverse applications in fundamental and applied research, for instance in gene therapy in medicine and disease control. More recent, a new hot point is to find a feasible way to use RNAi as an alternative method for practical application of crop protection to combat pest insects.

Chorion Genes: A Landscape of Their Evolution, Structure, and Regulation

Differential regulation at the level of transcription provides a means for controlling gene expression in eukaryotes, especially during development. Insect model systems have been extensively used to decipher the molecular basis of such regulatory cascades, and one of the oldest such model systems is the regulation of chorion gene expression during ovarian follicle maturation. Recent experimental and technological advances have shed new light onto the system, allowing us to revisit it. Thus, in this review we try to summarize almost 40 years worth of studies on chorion gene regulation while-by comparing Bombyx mori and Drosophila melanogaster models-attempting to present a comprehensive, unified model of the various regulatory aspects of choriogenesis that takes into account the evolutionary conservation and divergence of the underlying mechanisms.

Ecdysteroids and Ecdysteroid Signaling Pathways During Insect Oogenesis

During insect oogenesis, the oocyte acquires nutrients and genetic determinants to support embryonic development (previtellogenesis and vitellogenesis) and subsequently becomes surrounded by a protective eggshell (choriogenesis). In many insects, ecdysteroids are synthesized during ovarian growth which is often followed by the accumulation of ecdysteroid conjugates into the eggs. The exact role of the ecdysteroids during oogenesis remains largely unclarified although functions as paracrine or autocrine regulators to signal the progression of follicle development or the resumption of meiosis in the oocyte have been proposed. In the silkmoth, Bombyx mori, although ecdysteroids are synthesized by the ovarian follicles, progression of follicle development towards choriogenesis requires down-regulation in ecdysteroid signaling. Using the regulation of silkmoth oogenesis by 20-hydroxyecdysone as a starting point, this review discusses the physiological roles of ecdysteroids and the function of the ecdysone regulatory pathway during insect oogenesis.

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.

Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide.

Methoxyfenozide (RH-2485) is a non-steroidal ecdysteroid agonist with a dibenzoylhydrazine structure, representing a group used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ∼ 1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis also allows to gain insights into the mechanism of resistance and on the crosstalk between ecdysteroid signaling and cell proliferation-linked processes.

Saponins show high entomotoxicity by cell membrane permeation in Lepidoptera

BACKGROUNDnIn 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.nnnRESULTSnBoth 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.nnnCONCLUSIONSnThe 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.

Cell-Based Screening Systems for Insecticides

To date an average of ∼10 billion USD is spent per year for synthetic insecticides to control pest insects of importance in agriculture and human health. At early screening stages for novel insecticides and targets, there is an increasing interest in the development of in vitro methods to replace conventional animal toxicity tests. In this review we discuss the contributions of established insect cell lines, joined with high throughput screening procedures, to rapid screening of many synthetic and natural materials and accelerate the discovery of novel environmentally-safe control agents. Hence, we give significant recent examples and advances (e.g. approach with EcR reporter systems as a paradigm), and we offer a vision of the future of cell-based screening strategies.