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Insect Oogenesis

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Ecdysone signalling in the ovary of the silkmoth, Bombyx mori

In the silkmoth, Bombyx mori, oogenesis is initiated in the ovarian follicles at high titers of 20-hydroxy-ecdysone during pupal and pharate adult development. Studies using the ecdysone agonist tebufenozide have shown that the transition from vitellogenesis to choriogenesis requires down-regulation of ecdysone signalling. Expression studies and analysis of binding sites in promoter regions have lead to the identification of several putative regulatory factors in this pathway, including the orphan nuclear receptor BmE75 and the chorion gene regulator BmGATAβ.

 

Regulation of silkmoth oogenesis during pharate adult development by 20E. Panel A: Induction of vitellogenesis by rising titers of 20E in the hemolymph. Panel B: Induction of choriogenesis by declining titers of 20E in the hemolymph.

Swevers, L., and Iatrou, K. (2009).  In: Ecdysone, structures and fucntions (Smagghe, G., ed.), pp. 127-164. Springer-Verlag, Dordrecht, Nederland.

 

Recently, yeast two-hybrid screens using  BmE75C as bait were carried out to identify interacting partners and led to the isolation of BmCAP, a putative adaptor protein containing three SH3 domains at its C-terminus that is implicated in cytoskeleton organization, cell adhesion and insulin signalling in other developmental systems. Interestingly, BmCAP encodes multiple mRNA isoforms that can be processed in different protein isoforms. Localization studies indicate the presence of the large isoform, BmCAP-A, at the junctions of the cells of the follicular epithelium as well as at their apical surface during late choriogenesis, indicating functional roles in cell adhesion as well as in secretion of the chorion proteins and the sculpting of the egg shell surface. While a role for BmCAP-A in the modulation of the function of BmE75C was not found, an involvement for such a role may seem more likely for the short isoform, BmCAP-B.

Subcellular localization of BmCAP-A protein in the cells of the follicular epithelium during early and late choriogenesis by immunofluorescence. Shown are optical sections at different heights in the follicular epithelial cells: at the basal side, in the middle and at the apical side. At the basal side, BmCAP antibody stains the boundaries between the follicular cells. In the middle section, strong cytoplasmic staining is observed for the early choriogenic follicles while hardly any staining is present during late choriogenesis. At the apical side, the antibody stains the boundaries between the cells in early choriogenic follicles. In late choriogenic follicles, the staining resembles the typical surface sculpture of the eggshell during late choriogenesis.

Georgomanolis, T., Iatrou, K., and Swevers, L. (2009). Insect Biochem. Mol. Biol. 39, 892-902.

 

 

Insect Pest Control

Insect Growth Regulators

In Europe, more than 30,000 tonnes of insecticides are spread every year on field sites. Crop protection against insect pests involves alternatives to chemical treatments like the use of genetically modified organisms or biological control, but pesticides still remain the most efficient and cheap control strategy in most European countries.

Alternative methods of insect pest control include the use of insect growth regulators, which disrupt insect growth and development. Among these, diacylhydrazine (DAH) analogs, called ecdysone agonists or molting accelerating compounds, have been developed successfully for insect pest control. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects as does the natural insect molting hormone 20-hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects (caterpillar larvae of moths and butterflies), raising the question whether non-lepidopteran specific analogues can be isolated.

20E-responsive GFP reporter cell lines used to detect ecdysone mimetics. The cell lines are also used to monitor purification of active substances from plant extracts and to determine EC50 values for QSAR analysis, ligand–receptor modeling, and concentrations of active ecdysteroid in biological samples.

 

Swevers, L., Kravariti, L., Ciolfi, S., Xenou-Kokoletsi, M., Ragoussis, N., Smagghe, G., Nakagawa, Y., Mazomenos, B., and Iatrou., K. (2004). FASEB J. 18, 134-136.
To address this question, we have developed ecdysone-responsive lepidopteran, dipteran and coleopteran ecdysone-resonsive screening systems using Bm5 cells of Bombyx mori, S2 cells of Drosophila melanogaster and Ag3C cells of Anthonomus grandis, respectively, to discover and evaluate compounds that have order-specific ecdysone agonistic or antagonistic activity. Libraries of non-steroidal ecdysone agonists containing different mother structures, with DAH, acylaminoketon (AAK) and tetrahydroquinoline (THQ) analogues, as well as collections of plant extracts were tested. Our studies confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show such specificity, indicating that ecdysone agonist-based insecticides based on the THQ mother structure can be developed that are specific to other insect orders.

 

Environmental RNAi

RNA interference (RNAi) is a technique in which gene expression is silenced after introduction of dsRNAs of homologous sequences. Because of the ease of application, RNAi holds great promise for the genetic analysis of physiological and developmental processes in animals that are not readily amenable to genetic analysis. Moreover, because of its specificity, RNAi could be applied as a new means of environmentally friendly pest control.

A survey of the expression of genes that encode the core components of RNAi revealed remarkable differences between (i) a lepidopteran insect, the silkmoth Bombyx mori, the basis of the silk industry and an important model for the study of developmental processes in insects, and (ii) model representatives of dipteran and coleopteran insects, the fruitfly Drosophila melanogaster, and the flour beetle, Tribolium confusum, respectively, indicating considerable variability in efficiency of RNAi among insect groups.

Mechanisms of RNA silencing in Drosophila melanogaster: RNAi pathway and mRNA destruction.Bombyx mori tissues and Bm5 cells. Indicated is the dsRNA-binding protein R2D2 which has limited expression in

Buchon, N., and Vaury, C. (2006). Heredity 96, 195–202.

 

By comparing the process of RNAi among the three species, important inferences can be made to predict which factors may limit the effectiveness of RNAi in lepidopteran insects. These comparisons will extend at all levels at which RNAi efficiency can be affected such as the functioning of the RNAi core machinery, the uptake of dsRNA from the hemolymph or the food and the existence of dsRNA degrading enzymes in particular tissues or developmental stages. Factors whose expression is correlated with increased sensitivity to systemic RNAi will subsequently be tested in experiments of genetic transformation of tissue culture cell lines derived from lepidopteran insects for their capacity to improve the sensitivity of the cells to systemic RNAi.