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In the mosquito Aedes aegypti, blood feeding activates vitellogenesis that involves yolk protein precursor (YPP) genes in an insect metabolic tissue, the fat body. Vitellogenesis is regulated by the 20-hydroxyecdysone (20E) regulatory hierarchy, in which the Ets-domain protein E74 is a key transcriptional regulator. The mosquito AaE74 gene encodes two isoforms-AaE74A and AaE74B. Both AaE74 isoforms are 20E-inducible early gene products. AaE74B reaches its maximal expression at 10(-7)M of 20E, while AaE74A requires 10(-6)M of 20E, a concentration at which the YPP genes reach their maximal induction level. In transfection assay, AaE74B is capable of activating a reporter construct containing E74-response elements, while expression of AaE74A has no effect on the basal levels of the reporter. The AaE74B binding activity is present in the fat body nuclei only during active vitellogenesis. Taken together, our findings demonstrate that AaE74B isoform plays the role of a transcriptional activator during vitellogenesis.

In mosquitoes, the steroid 20-hydroxyecdysone (20E) is the main regulator of yolk protein precursor (YPP) gene expression. However, peptide hormones have also been implicated. To investigate involvement of the cAMP-mediated signal-transduction cascade in regulation of mosquito vitellogenic events, we cloned an Aedes aegypti cAMP response element binding protein (AaCREB). The AaCREB contained the domains characteristic to members of the cAMP response element binding protein (CREB) family of transcription factors: a kinase inducible domain region and a bZIP domain responsible for DNA binding and protein dimerization. In the mosquito fat body (site of YPP gene expression), the AaCREB gene was constitutively expressed and produced a transcript of 3.5-4 kb. In vitro fat body organ culture experiments demonstrated that elicitors of the cAMP signal-transduction pathway attenuated 20E-stimulated YPP gene expression. Cell transfection analysis indicated that AaCREB served as a potent repressor of transcription (designated AaCREBr). The role of AaCREBr as a transcriptional repressor supported the electrophoretic mobility shift assay (EMSA) with nuclear extracts from vitellogenic fat bodies. This analysis detected CREB-specific band-shift complexes in nuclear extracts at 24 and 36 h post-blood meal (PBM), when YPP gene expression reaches its peak then terminates. Examination of the regulatory regions of two major YPP genes, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP), revealed the presence of putative CREB response elements (CREs). These elements competed with the CRE consensus sequence for binding of in vitro-expressed AaCREBr. We propose that AaCREBr functions as a repressor of YPP gene expression at the time of vitellogenesis termination in the fat body.

In the anautogenous mosquito, Aedes aegypti, vitellogenesis is under the strict control of 20-hydroxyecdysone (20E), which is produced via a blood meal-activated hormonal cascade. Several genes of the ecdysteroid-regulatory hierarchy are conserved between vitellogenesis in mosquitoes and metamorphosis in Drosophila. We report characterization of two isoforms of the mosquito early E74 gene (AaE74), which have a common C-terminal Ets DNA-binding domain and unique N-termini. They exhibited a high level of identity to Drosophila E74 isoforms A and B and showed structural features typical for Ets transcription factors. Both mosquito E74 isoforms bound to an E74 consensus motif C/AGGAA. In the fat body and ovary, the transcript of AaE74 isoform homologous to Drosophila E74B was induced by a blood meal exhibiting its highest level coinciding with the peak of vitellogenesis. In contrast, the transcript of AaE74 isoform homologous to Drosophila E74A was activated at the termination of vitellogenesis. These findings suggest that AaE74A and AaE74B isoforms play different roles in regulation of vitellogenesis in mosquitoes.

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10(-7) or 10(-8) M), partially inhibited the response to JH III.

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes which encode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. Here, we identify the homologue of the Drosophila E75 early ecdysone inducible gene in the yellow fever mosquito Aedes aegypti, and show that, as in Drosophila, the mosquito homologue, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses.