Marianna Zinovjevna Kapitskaya

The mosquito ultraspiracle homologue, a partner of ecdysteroid receptor heterodimer: cloning and characterization of isoforms expressed during vitellogenesis.

We report the cloning and characterization of two isoforms of the Ultraspiracle homologue (AaUSP) from the mosquito, Aedes aegypti. The 2.33-kb AaUSPa cDNA has an open reading frame (ORF) of 484 amino acids encoding a polypeptide of 54 kDa, whereas the 2.14-kb AaUSPb ORF of 459 amino acids encodes a 51.3 kDa polypeptide. The AaUSPa and AaUSPb proteins differ only in the N-terminal portion of the variable A/B domain. The AaUSP DNA-binding domain shares 92% and 97% identities with the respective domains of the Drosophila (DmUSP) and Bombyx (BmUSP) Ultraspiracles. However, the AaUSP ligand-binding domain is only 57% and 52% identical to those of DmUSP and BmUSP, respectively. In spite of the relatively low level of sequence conservation, electrophoretic mobility shift assay (EMSA) and hormone-binding assay clearly demonstrated that the products of the AaUSPa and AaUSPb cDNAs are functional heterodimeric partners of the mosquito ecdysteroid receptor. In vitellogenic tissues, each of the two AaUSP isoforms is expressed differently: the AaUSPa is predominant in the fat body and the AaUSPb in the ovary. The kinetics of ovarian AaUSP mRNA coincide with those of the ecdysteroid receptor, being elevated during the previtellogenic period and shortly after the onset of vitellogenesis. In contrast, the level of the AaUSP in the fat body remains relatively constant throughout most of the vitellogenic cycle.

Mosquito ecdysteroid receptor: Analysis of the cDNA and expression during vitellogenesis

An insect steroid hormone, 20-hydroxyecdysone (20E), plays an important role in regulating egg maturation in mosquitoes. To better understand its role, we cloned the cDNA coding for the putative ecdysteroid receptor from the mosquito, Aedes aegypti (AaEcR). The 4158 bp AaEcR cDNA has an open reading frame of 675 amino acids with 10 potential glycosylation sites and a putative phosphorylation polyserine domain. The AaEcR has a DNA binding domain with two zinc fingers and a ligand binding domain characteristic of members of the steroid hormone receptor superfamily. These AaEcR domains share 97 and 87% identities with the respective domains of the Drosophila ecdysteroid receptor (DmEcR). However, the A/B region of the AaEcR shares 35% identity with that of DmEcR-B1 isoform. The F region, located at the carboxyl-terminal of the AaEcR, has only 9% identity with the corresponding region of DmEcR. Potential nuclear targeting and dimerization signals are also present in the AaEcR sequence. There are three AaEcR transcripts of 4.2 kb, 6 kb and 11 kb in adult mosquitoes. 4.2 kb mRNA is predominantly expressed in female mosquitoes during vitellogenesis. In both the fat body and ovaries of the female mosquito, the level of AaEcR mRNA is high at the previtellogenic period and after the onset of vitellogenesis (6 h post blood meal, PBM).

Expression of the early-late gene encoding the nuclear receptor HR3 suggests its involvement in regulating the vitellogenic response to ecdysone in the adult mosquito

The insect steroid hormone, 20-hydroxyecdysone (20E), is a key factor controlling critical developmental events of embryogenesis, larval molting, metamorphosis, and, in some insects, reproduction. We are interested in understanding the molecular basis of the steroid hormone ecdysone action in insect egg development. The yellow fever mosquito, Aedes aegypti, in addition to being an important vector of human diseases, represents an outstanding model for studying molecular mechanisms underlying egg maturation due to stringently controlled, blood meal-activated reproductive events in this insect. To elucidate the genetic regulatory hierarchy controlling the reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. We have previously demonstrated the conservation of a primary ecdysone-triggered regulatory hierarchy, implicated in development of immature stages of Drosophila, represented by the ecdysone receptor/Ultraspiracle complex and an early gene E75 during the reproductive ecdysone response (Wang, S.-F., Miura, K., Miksicek, R.J., Segraves, W.A., Raikhel, A.S., 1998. DNA binding and transactivation characteristics of the mosquito ecdysone receptor - Ultraspiracle complex. J. Biol. Chem. 273, 27531-27540; Pierceall, W. E., Li, C., Biran, A., Miura, K., Raikhel, A.S., Segraves, W.A., 1999. E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction. Mol. Cell. Endocrinol. 150, 73-89). The present paper demonstrates that conservation of the factors involved in the ecdysone-responsive genetic hierarchy regulating female reproduction extends beyond the early genes. Here, we identify AHR3, a highly conserved homologue of the Drosophila HR3 early-late ecdysone-inducible gene in the mosquito. We show that AHR3 is expressed in both vitellogenic tissues of the female mosquito, the fat body and the ovary. The expression of AHR3 correlates with the ecdysteroid titer, reaching a peak at 24 h after a blood meal. Moreover, in vitro fat body culture experiments demonstrate that the kinetics and dose response of AHR3 to 20-hydroxyecdysone (20E), an active ecdysteroid in the mosquito, is similar to those of the late vitellogenic genes rather than the early E75 gene. However, as shown for other early and early-late genes, the 20E activation of AHR3 is not inhibited by the presence of cycloheximide, a protein synthesis inhibitor. Taken together, these findings strongly suggest AHR3 involvement in regulating the vitellogenic response to ecdysone in the adult mosquito.