Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones.

Many plant compounds are able to modulate growth and reproduction of herbivores by directly interacting with steroid hormone systems. In insects, several classes of phytochemicals, including the phytoestrogens, interfere with molting and reproduction. We investigated whether the anti-ecdysone activity may be due to interaction with the ecdysone receptor (EcR) using a reporter-gene assay and a cell differentiation assay of an ecdysone-responsive cell line, Cl.8+. We tested rutin (delays molt in insects); four flavones: luteolin and quercetin (metabolites of rutin), and apigenin and chrysin; and three non-flavones, coumestrol and genistein (both estrogenic) and tomatine (alters molt in insects). None of the phytochemicals tested were ecdysone agonists in the reporter-gene assay, but the flavones were able to significantly inhibit EcR-dependent gene transcription. In the Cl.8+ cells, quercetin and coumestrol were mixed agonists/antagonists, while genistein, tomatine and apigenin showed a synergistic effect with ecdysteroid in the reduction of cell growth. We suggest that the rutin effects on molting in insects are most likely due to the metabolites, luteolin or quercetin, while tomatine acts via a non-EcR pathway. Flavones not only interact with EcR and estrogen receptor (ER), but also signal nitrogen-fixing bacteria to form root nodules. The NodD protein which regulates this symbiosis has two ligand-binding domains similar to human ERalpha. The evolutionary significance of these findings are discussed.

Adhesion of Drosophila imaginal disc cells in vitro.

We have used our imaginal disc cell lines to carry out in vitro studies on the cell-cell and cell-substrate adhesion of Drosophila leg and wing disc cells. Single cells were allowed to reaggregate in roller culture, and this process was found to be partially dependent on the presence of magnesium and calcium ions in the suspension medium. Varying rates of reaggregation were observed in cells from different stages of a passage, correlating with the pattern of morphogenesis which occurs during the passage. We have demonstrated that cloned cell lines can be produced showing certain selected characteristics, such as reduced cell adhesiveness.

Cell-cell and cell-substrate adhesion in cultured Drosophila imaginal disc cells.

Drosophila imaginal disc cell lines were used to investigate various aspects of cellular adhesion in vitro. The distribution of PS integrins and their involvement in cell-cell and cell-substrate adhesion were assessed with the monoclonal antibody aBG-1 against the betaPS subunit, and both forms of adhesion were found to be impeded by the presence of the antibody. Adhesion to a number of extracellular matrix components was investigated, and the cells were found to adhere to human fibronectin. This adhesion was inhibited by aBG-1. The adhesion molecule fasciclin III was also found in these cells. Given that the cells are competent to perform cell-cell and cell-substrate adhesion, it was thought that apical basal polarity might be restored when other suitable conditions were provided, i.e., an artificial basement layer with feeder cells to provide nutrients basally to the cells, and some features of apical-basal morphology were seen in cells cultured under these conditions.

Three subfamilies of pheromone and receptor genes generate multiple B mating specificities in the mushroom Coprinus cinereus.

The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.

Interaction of PAHs and PCBs with ecdysone-dependent gene expression and cell proliferation.

This study was done to determine whether PAHs and PCBs can interact with the arthropod steroid hormone system. Ecdysteroid molting hormones control growth, molting, and reproduction in arthropods. A spike in 20-OH ecdysone (20 HE) triggers the molt cycle in crustaceans, and earlier studies have shown that PAHs can affect this molt cycle in several crab species. However, the mechanism of this molt cycle interaction is unknown. Both PAHs and PCBs interact with other nuclear receptors; however, nothing is known about their ability to interact with the invertebrate ecdysone receptor (EcR). Four PAHs, benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and chrysene, and the commercial PCB mixture, Aroclor 1254, were used to determine the ability of these classes of compounds to induce ecdysone-dependent reporter gene expression and to modify the proliferation and differentiation response of the ecdysteroid-responsive Cl.8+ cell line. The four PAHs were each able to enhance the ecdysteroid response in both the reporter gene and the cell proliferation assays only when given in conjunction with ecdysteroids. Aroclor 1254 had no effect in either system, either alone or in conjunction with ecdysteroids. These studies show that although the PAHs alone do not activate ecdysteroid-dependent gene expression or cell differentiation, they are able to enhance the effect of ecdysteroids, presumably through a non-receptor-mediated process. This mechanism may explain the effects on molting which have been reported after low-level crude oil exposures in crustaceans.

The effect of juvenile hormone on the response of the Drosophila imaginal disc cell line Cl 8+ to moulting hormone.

The Drosophila wing imaginal disc cell line Cl 8+ was used to investigate the interaction between juvenile hormone III (JH) and 20-hydroxyecdysone (20HE). Cell cultures were exposed to either or both hormones at a range of concentrations and cell growth was observed. JH was found to ameliorate the effects of 20HE on cell growth, even when added after the cells had been exposed to 20HE for 4 or 24h.

The effects of several ecdysteroids and ecdysteroid agonists on two Drosophila imaginal disc cell lines.

Two Drosophila imaginal disc cell lines, C18+ (sensitive to 20-hydroxyecdysone, 20HE) and C18R (resistant to 20HE) were exposed to the ecdysteroid agonists RH5849 and RH5992 and the ecdysteroids inokosterone, makisterone A and muristerone A. All compounds tested were found to have similar effects on the cells, comparable to the effects of 20HE, although at different concentrations. C18R showed resistance to all compounds, again at varying concentrations. We conclude that it is likely that all the compounds tested use the same receptors as 20HE, but show maximum effectiveness at different concentrations.

Effect of age on the growth and response of a Drosophila cell line to moulting hormone.

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line Cl8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of beta-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.

Parameters of growth in primary cultures and cell lines established from Drosophila imaginal discs.

We have further characterised our tissue culture system for the growth in vitro of Drosophila imaginal disc cells, including the culture medium requirements for optimum growth and we have adjusted the protocol recommended for the initiation of cultures. Many imaginal disc fragments become organised into vesicles, and some of these secrete extracellular material into the lumen. Sensory axons differentiate in primary disc cultures, in the absence of bristle formation. The early stages of cell division to form a cell line are recorded.

Cuticle secretion in Drosophila wing imaginal discs in vitro: parameters of exposure to 20-hydroxy ecdysone.

We have cultured Drosophila wing imaginal discs in vitro under a variety of hormonal conditions in order to determine whether cuticle secretion is enhanced by a withdrawal of 20-hydroxy ecdysone at one of two points in development, corresponding to the drop in hormone titer during the prepupal period, and to the fall in hormone levels during the later stages if imaginal differentiation. We found that these treatments did not enhance either pupal or adult cuticle secretion.

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines.

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.

The final stages of wing development in Drosophila melanogaster.

A brief description is given of the behavioural changes in Drosophila melanogaster at the time of eclosion and wing expansion. The developmental changes in the wing during this period are classified into four stages (0-III). Details of changes in the morphology of both the cuticle and the wing epidermal cells are given using both light and electron microscopy. It was found that the breakdown of the wing epidermis is initiated before wing expansion starts. The results obtained for Drosophila are compared with those for other insect species.

The eversion and differentiation of Drosophila melanogaster leg and wing imaginal discs cultured in vitro with an optimal concentration of beta-ecdysone.

The stages of the eversion and differentiation of prothoracic leg and wing discs of Drosophila melanogaster, in Shields and Sang's medium 3, are described. The range of specific imaginal structures produced, including patterns of sensilla trichodea and sensilla campaniformia, are noted, and the relationship of these structures to those differentiated in situ is discussed.

Active ion transport and beta-acdysone induced differentiation of Drosophila melanogaster imaginal discs cultured in vitro.

The theory that beta-ecdysone initiates developmental changes during insect metamorphosis by causing an increase in intranuclear levels of potassium, together with a concomitant decrease in sodium levels, has been investigated by two methods. First, imaginal discs from late third instar larvae have been cultured with 0-2 microng/ml of beta-ecdysone together with inhibitors of active ion transport. Non-specific inhibitors, which may have general effects on sulphydryl groups, such as iodoacetic acid, N-ethylmaleimide ethacrinic acid and furosemide, inhibit both eversion and differentiation at concentrations of from 10(-3) M to 2 X 10(-3) M. Ouabain, the only specific inhibitor of the active transport of Na+ and K+ across membranes, had no effect on development even at a concentration of 10(-2) M. Second, a medium containing raised levels of K+, and reduced concentrations of Na+, neither initiated disc development in the absence of beta-ecdysone, nor stimulated development induced by suboptimal levels (0-02 microng/ml) of beta-ecdysone, either in the presence or absence of ouabain. These results suggest that beta-ecdysone induced morphogenesis is not dependent upon Na+ and K+ concentrations, or on the activity of an ouabain-sensitive ion pump.