Ecdysteroid titers and expression profiles of ecdysone-regulated genes were determined during the last instar larval and during the pupal stages of Aedes aegypti (Diptera: Culicidae). Three peaks of ecdysteroids occurring at approximately 24, 30-33 and 45-48h after ecdysis to the fourth instar larval stage were detected. In the pupa, a large peak of ecdysteroids occurred between 6 and 12h after ecdysis to the pupal stage. A small rise in ecdysteroids was also detected at the end of the pupal stage. Quantitative reverse transcriptase polymerase chain reaction analyses of the expression of ecdysone receptors and ecdysone-regulated genes showed that the peaks of expression of most of these genes coincided with the rise in ecdysteroid levels during the last larval and pupal stages. In the last larval stage, ecdysteroid titers and mRNA expression profiles of ecdysone-regulated genes are similar to those observed for Drosophila melanogaster. However, in the early pupal stage, both ecdysteroid titers and the expression of ecdysone-regulated genes are somewhat different from those observed in D. melanogaster, probably because the duration of the pupal stage in D. melanogaster is 84h while in Ae. aeqypti the duration is only 48h. These data which describe the relationship between ecdysteroid titers and mRNA levels of Ae. aegypti ecdysteroid-regulated genes lay a solid foundation for future studies on the hormonal regulation of development in mosquitoes.
AB - Investigations of ecdysteroid-regulated gene cascades in Drosophila have shown that characteristics of downstream genes in such cascades include their repression by high ecdysteroid levels, their expression at low hormone levels, and the dependence of their expression on protein synthesis. In an earlier study, we identified a gene, esr20, which is expressed in the tracheae of the tobacco hornworm, Manduca sexta, prior to larval and pupal ecdyses. Initial characterization of the expression of esr20 suggested that it had the above characteristics of a downstream gene in an ecdysteroid-regulated cascade. The present study shows that, unlike the downstream genes in Drosophila, the expression of esr20 in tracheae cannot be induced by changes in the ecdysteroid levels alone. We present evidence which suggests that a decline in ecdysteroid is necessary but not sufficient for expression. Soon after pupal ecdysis the level of the esr20 transcript drops fourfold, and by 24 h after ecdysis the transcript is undetectable. Evidence is presented which suggests that this decline in transcript levels requires protein synthesis and appears to result from a decline in the stability of the transcript.