A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti.

In the mosquito Aedes aegypti, vitellogenesis is activated via an ecdysteroid hormonal cascade initiated by a blood meal. The functional ecdysone receptor is a heterodimer composed of the ecdysone receptor (EcR) and ultraspiracle, the homolog of the retinoid X receptor. The precise tuning of this hormonal response requires participation of both positive and negative transcriptional regulators. In Drosophila, Svp, a homolog of chicken ovalbumin upstream promoter transcription factor (COUP-TF), inhibits ecdysone receptor complex-mediated transactivation in vitro and in vivo. Here we report the cloning and characterization of the Svp homolog in mosquito Aedes aegypti, AaSvp. It possesses a high degree of amino acid sequence similarity to the members of the COUP-TF/Svp subfamily. AaSvp transcripts and protein are present in the fat body at high levels from the state of arrest to about 60 h post blood meal. AaSvp binds strongly to a variety of direct repeats of the sequence AGGTCA, but weakly to inverted repeats such as hsp27 EcRE. Transient transfection assays in Drosophila S2 cells showed that AaSvp was able to repress 20-hydroxyecdysone (20E)-dependent transactivation mediated by the mosquito ecdysteroid receptor complex. These data suggest that AaSvp negatively regulates the 20E signaling in the fat body during mosquito vitellogenesis.

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti.

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.

Three isoforms of a hepatocyte nuclear factor-4 transcription factor with tissue- and stage-specific expression in the adult mosquito.

We cloned three isoforms of hepatocyte nuclear factor-4 (HNF-4) from the mosquito Aedes aegypti, designated AaHNF-4a, AaHNF-4b, and AaHNF-4c. AaHNF-4a and AaHNF-4b are typical members of the HNF-4 subfamily of nuclear receptors with high amino acid conservation. They differ in N-terminal regions and exhibit distinct developmental profiles in the female mosquito fat body, a metabolic tissue functionally analogous to the vertebrate liver. The AaHNF-4b mRNA is predominant during the previtellogenic and vitellogenic phases, while the AaHNF-4a mRNA is predominant during the termination phase of vitellogenesis, coinciding with the onset of lipogenesis. The third isoform, AaHNF-4c, lacks part of the A/B and the entire C (DNA-binding) domains. The AaHNF-4c transcript found in the fat body during the termination of vitellogenesis may serve as a transcriptional inhibitor. Both AaHNF-4a and AaHNF-4b bind to the cognate DNA recognition site in electrophoretic mobility shift assay. Dimerization of AaHNF-4c with other mosquito HNF-4 isoforms or with mammalian HNF-4 prevents binding to the HNF-4 response element. In transfected human 293T cells, AaHNF-4c significantly reduced the transactivating effect of the human HNF-4alpha1 on the apolipoprotein CIII promoter. Electrophoretic mobility shift assay confirmed the presence of HNF-4 binding sites upstream of A. aegypti vg and vcp, two yolk protein genes expressed in the female mosquito fat body during vitellogenesis. Therefore, HNF-4, an important regulator of liver-specific genes, plays a critical role in the insect fat body.

Analysis of the mosquito lysosomal aspartic protease gene: an insect housekeeping gene with fat body-enhanced expression.

In the fat body of insects with cyclic egg maturation, lysosomes play a critical role in the termination of vitellogenesis by selectively degrading the secretory machinery involved in the massive production of yolk protein precursors. To investigate this fat body-specific lysosomal activity in the mosquito, a cathepsin D-like aspartic protease (LAP) was previously purified and its cDNA cloned. Here we report the isolation of the AaLAP gene from an Aedes aegypti genomic library. The transcribed region of the gene is comprised of five exons, spanning 1904 base pairs. Restriction fragment length polymorphism (RFLP) and genomic clone analyses show this gene to be single copy and polymorphic. Primer extension analysis revealed two putative transcription start sites (TSS). The extension products corresponding to the distal and proximal TSSs were present in both pre- and vitellogenic fat bodies, suggesting that both TSSs are involved in housekeeping as well as tissue-specific expression of this gene. TATA box-like and arthropod initiator sequences, hallmarks of regulated genes, were present near each putative TSS. Several sequences resembling binding sites for liver- and fat body-specific transcription factors were identified within 1 kb upstream and downstream of the gene. Significantly, direct binding for the C/EBP and GATA families of transcription factors was demonstrated in vitro by electrophoretic mobility shift assays (EMSA). Three sequences located upstream of AaLAP resembled the Drosophila melanogaster yolk protein fat body enhancer (Dm Yp FBE). Potential hormone-response elements were also recognized in the gene; however, they did not bind the mosquito ecdysteroid receptor/Ultraspiracle heterodimer in EMSA experiments, indicating that these sequences may interact with different nuclear receptors.