Activation of Aedes aegypti prophenoloxidase-3 and its role in the immune response against entomopathogenic fungi.

Serine protease cascade-mediated melanization is an important innate immune response in insects and crustaceans, which involves the proteolytic activation of prophenoloxidase (PPO). In this study, we investigated the role of Aedes aegypti PPO3 in antifungal immune defence. We expressed and purified recombinant PPO3 (rPPO3) in Escherichia coli and demonstrated that rPPO3 was activated by ethanol and, to a lesser extent, by cetylpyridinium chloride. In the presence of Cu2+ , rPPO3 exhibited enzyme activity. Immunoblot results revealed that the rPPO3 was cleaved by the haemolymph from immune-challenged mosquitoes or purified Ostrinia furnacalis serine protease 105 in vitro. The cleaved rPPO3 converted dopamine to toxic intermediates that killed fungal conidia of Beauveria bassiana in vitro. In mosquitoes challenged with Be. bassiana, cleavage of rPPO3 produced a 50 kDa phenoloxidase (PO) fragment. Further analysis revealed that the survival rate of mosquitoes with fungal infection increased significantly following injection of rPPO3 into the haemocoel. Taken together, our results suggest that proteolytic cleavage of the mosquito PPO3 plays an important role in the antifungal immune response. This has led to a better understanding of the mechanism of PPO activation in the mosquito and the role of melanization in the antifungal immune response.

The sex locus is tightly linked to factors conferring sex-specific lethal effects in the mosquito Aedes aegypti.

In many taxa, sex chromosomes are heteromorphic and largely non-recombining. Evolutionary models predict that spread of recombination suppression on the Y chromosome is fueled by the accumulation of sexually antagonistic alleles in close linkage to the sex determination region. However, empirical evidence for the existence of sexually antagonistic alleles is scarce. In the mosquito Aedes aegypti, the sex-determining chromosomes are homomorphic. The region of suppressed recombination, which surrounds the male-specific sex-determining gene, remains very small, despite ancient origin of the sex chromosomes in the Aedes lineage. We conducted a genetic analysis of the A. aegypti chromosome region tightly linked to the sex locus. We used a strain with an enhanced green fluorescent protein (EGFP)-tagged transgene inserted near the male-determining gene to monitor crossing-over events close to the boundary of the sex-determining region (SDR), and to trace the inheritance pattern of the transgene in relation to sex. In a series of crossing experiments involving individuals with a recombinant sex chromosome we found developmental abnormalities leading to 1:2 sex biases, caused by lethality of half of the male or female progeny. Our results suggest that various factors causing sex-specific lethal effects are clustered within the neighborhood of the SDR, which in the affected sex are likely lost or gained through recombination, leading to death. These may include genes that are recessive lethal, vital for development and/or sexually antagonistic. The sex chromosome fragment in question represents a fascinating test case for the analysis of processes that shape stable boundaries of a non-recombining region.

The RNA-Seq approach to studying the expression of mosquito mitochondrial genes.

In this study, we used extensive expressed sequence tag evidence obtained through 454 and Solexa next-generation sequencing to explore mtDNA transcription in male and female first instar larvae of Aedes aegypti and adults of Aedes aegypti, Anopheles gambiae, and Anopheles quadrimaculatus. Relative abundances of individual transcripts differed considerably within each sample, consistent with the differential stability of messenger RNA species. Large differences were also observed between species and between larval and adult stages; however, the male and female larval samples were remarkably similar. Quantitative PCR analysis of selected genes, cox1, l-rRNA and nd5, in larvae and adults of Ae. aegypti and in An. gambiae adults was consistent with the RNA-Seq-based quantification of expression. Finally, the absence of a conserved mtDNA region involved in transcriptional control in other dipterans suggests that mosquitoes have evolved a distinct mechanism of regulation of gene expression in the mitochondrion.

The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti.

The broad (br) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5'-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.

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.

Transcriptional regulation of the mosquito vitellogenin gene via a blood meal-triggered cascade.

In anautogenous mosquitoes, a blood meal is required for activation of genes encoding yolk protein precursors (YPP). Vitellogenin (Vg), the major YPP gene, is transcribed at a very high level following blood meal activation. It is expressed exclusively in the female fat body, the tissue producing most of mosquito hemolymph and immune proteins. In this paper, we analyzed the upstream region of the Aedes aegypti Vg gene in order to identify regulatory elements responsible for its unique expression pattern. To achieve this goal, we analyzed the gene using transgenic Drosophila and Aedes as well as DNA-binding assays. These analyses revealed three regulatory regions in the 2.1 kb upstream portion of the Vg gene. The proximal region containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh is required for the correct tissue- and stage-specific expression at a low level. The median region carrying sites for early ecdysone response factors E74 and E75 is responsible for hormonal enhancement of Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic of the Vg gene. The present work elucidates the molecular basis of blood meal-dependent expression of this mosquito gene, laying the foundation for mosquito-specific expression cassettes with predictable stage and tissue specificity.

Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].

We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.

Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti.

Lipophorin (Lp) functions as a yolk protein precursor in the mosquito Aedes aegypti and it is internalized via receptor-mediated endocytosis (Insect Biochem. Mol. Biol., 30 (2000) 1161). We cloned and molecularly characterized a putative mosquito ovarian lipophorin receptor (AaLpRov) cDNA. The cDNA has a length of 3468 bp coding for a 1156-residue protein with a predicted molecular mass of 128.9 kDa. The deduced amino acid sequence of the cDNA revealed that it encodes a protein homolog of the LDL receptor superfamily, and that it harbors eight cysteine-rich ligand binding repeats at the N-terminus like vertebrate VLDL receptors. The deduced amino acid sequence of this mosquito ovarian receptor is most similar to that of the locust lipophorin receptor (LmLpR) (64.3%), and is only distantly related to the mosquito vitellogenin receptor (VgR) (18.3%), another ovarian LDLR homolog with a different ligand. The AaLpRov cDNA was expressed in a TnT Coupled Reticulocyte Lysate system, and co-immunoprecipitation experiments confirmed that the receptor protein specifically binds Lp. Developmental expression profiles clearly showed that AaLpRov transcripts are present in the vitellogenic ovary, with peak expression at 24-36 h post blood meal. In situ hybridization indicated that AaLpRov transcripts are present only in female germ line cells. Distance-based phylogenetic analyses suggest that the insect LpR and vertebrate LDL/VLDL receptor lineages separated after divergence from the insect VgR lineage.

A novel function of 20-hydroxyecdysone: translational repression of the lysosomal protease mRNA in the mosquito fat body.

In the female fat body of the mosquito Aedes aegypti, lysosomes play important roles during the cessation of vitellogenesis by degrading the biosynthetic machinery and aiding the remodeling of the fat body cells. A detailed study of a mosquito lysosomal aspartic protease (AaLAP) has shown a unique expression pattern in the vitellogenic fat body: the level of AaLAP mRNA dramatically rises and peaks at 24 h post blood meal (PBM) correlating with the high titer of ecdysteroids; however, there is a 12 h lag before peak levels of AaLAP protein and its enzymatic activity has been observed. These observations suggest that the high titer of 20-hydroxyecdysone (20E) may hinder translation of the AaLAP mRNA. Here, we used an in vitro organ culture to study the effect of 20E on the protein synthesis of AaLAP in the fat body. The increase in the AaLAP protein level in the fat body, dissected at 24 h PBM and incubated for 6 or 12 h, was inhibited by the presence of 10(-5) M 20E in the medium. Incubation in the hormone-free medium did not effect accumulation of the AaLAP protein which proceeded at the levels comparable to the intact insect. Furthermore, the effect of 10(-5) M 20E on the AaLAP accumulation was reversible. These experiments support the hypothesis of the 20E-mediated repression of lysosomal protease mRNAs at the translational level in the regulation of vitellogenic and postvitellogenic events in the mosquito fat body. Analysis of the 5' and 3' -end untranslated regions (UTR) of AaLAP mRNA form secondary structures suggest that they may also contribute to mRNA stability and 20E-mediated translational inhibition.

The vitellogenin gene of the mosquito Aedes aegypti is a direct target of ecdysteroid receptor.

In the female mosquito Aedes aegypti, vitellogenin (Vg), the major YPP, is activated by 20-hydroxyecdysone (20E) at the transcriptional level. We used cell transfection assays in the Drosophila S2 cells to investigate whether 20E acts directly on the Vg gene via its functional receptor, the heterodimer composed of the ecdysteroid receptor (EcR) and the ultraspiracle (USP) proteins. We demonstrated that the Vg 5'-regulatory region contains a functional ecdysteroid-responsive element (VgEcRE1) that is necessary to confer responsiveness to 20E. VgEcRE binds directly to EcR-USP produced in vitro and extracted from the vitellogenic fat body nuclei. The binding intensity of the EcR-USP-EcRE1 complex from nuclear extracts corresponds to the levels of ecdysteroids and of the Vg transcript during the vitellogenic cycle. Given the modest level of 20E-dependent activation, it is likely that the EcR-USP receptor acts synergistically with other transcription factors to bring about the high level of Vg gene expression.

A novel GATA factor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with the CtBP corepressor.

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of many devastating human diseases. An important adaptation for anautogenicity is the previtellogenic arrest (the state of arrest) preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the upstream region of the YPP genes serves as a transcriptional repressor at the state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female fat body. Furthermore, in nuclear extracts of previtellogenic fat bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of AaGATAr. The specific repression of YPP genes by AaGATAr in the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity.

Organization and developmental expression of the mosquito vitellogenin receptor gene.

Vitellogenin is a precursor of the major yolk protein, vitellin. It is internalized by developing oocytes via receptor-mediated endocytosis. Previously, we characterized the vitellogenin receptor (VgR) from oocytes of the mosquito Aedes aegypti [Sappington, T.W., Kokoza,V.A., Cho,W.L. and Raikhel,A.S. (1996) Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor. Proc Natl Acad Sci USA 93: 8934-8939]. The VgR receptor has a unique structure with two putative ligand-binding domains. In order to understand the regulation of this important molecule, we characterized the VgR gene structure and its expression during vitellogenesis in the mosquito A. aegypti. We report here that the VgR gene was separated by five introns that have an average length of 60 bp, except for the second intron which was more than 20 kb long. Most introns were located within the coding regions of the first protein domain. We isolated two allelic variations of the VgR gene, VgR1 and VgR2, the nucleotide sequences of which differing only in their 5'-flanking regions. Considering their frequency in the mosquito genome, VgR2 appeared to be a major allele. The expression of VgR mRNA was studied by the Northern blot analysis and in situ hybridization. The level of the VgR transcript started to rise in the ovary one day post-eclosion. It continued its dramatic rise during the vitellogenic period, reaching its peak at 24 h PBM. The VgR transcript was present exclusively in ovaries where it was seen in oocytes and nurse cells of primary follicles and germ-line cells of the germarium.

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.

Conserved molecular mechanism for the stage specificity of the mosquito vitellogenic response to ecdysone.

In the mosquito Aedes aegypti, the adult female becomes competent for a vitellogenic response to ecdysone after previtellogenic development. Here, we show that betaFTZ-F1, the nuclear receptor implicated as a competence factor for stage-specific responses to ecdysone during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. AaFTZ-F1 is expressed highly in the mosquito fat body during pre- and postvitellogenic periods when ecdysteroid titers are low. The mosquito AaFTZ-F1 transcript nearly disappears in mid-vitellogenesis when ecdysteroid titers are high. An expression peak of HR3, a nuclear receptor implicated in the activation of betaFTZ-F1 in Drosophila, precedes each rise in mosquito FTZ-F1 expression. In in vitro fat body culture, AaFTZ-F1 expression is inhibited by 20-hydroxyecdysone (20E) and superactivated by its withdrawal. Following in vitro AaFTZ-F1 superactivation, a secondary 20E challenge results in superinduction of the early AaE75 gene and the late target VCP gene. Electrophoretic mobility-shift assays show that the onset of ecdysone-response competence in the mosquito fat body is correlated with the appearance of the functional AaFTZ-F1 protein at the end of the previtellogenic development. These findings suggest that a conserved molecular mechanism for controlling stage specificity is reiteratively used during metamorphic and reproductive responses to ecdysone.

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors.

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

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.

Differential expression and regulation by 20-hydroxyecdysone of mosquito ultraspiracle isoforms.

Ultraspiracle (USP), the insect homologue of the vertebrate retinoid X receptor, is an obligatory dimerization partner for the ecdysteroid receptor (EcR). Two USP isoforms, USP-A and USP-B, with distinct N-termini, occur in the mosquito Aedes aegypti. In the fat body and ovary, USP-A mRNA is highly expressed during the pre- and late vitellogenic stages, corresponding to a period of low ecdysteroid titer, while USP-B mRNA exhibits its highest levels during the vitellogenic period, correlating with a high ecdysteroid titer. Remarkably, 20-hydroxyecdysone (20E) has opposite effects on USP isoform transcripts in in vitro fat body culture. This steroid hormone upregulates USP-B transcription and its presence is required to sustain a high level of USP-B expression. In contrast, 20E inhibits activation of USP-A transcription. Although EcR.USP-A recognizes the same ecdysteroid-responsive elements, EcR.USP-B binds them with an affinity twofold higher than that of EcR.USP-A. Likewise, EcR.USP-B transactivates a reporter gene in CV-1 cells twofold more strongly than EcR.USP-A. These results suggest that USP-B functions as a major heterodimerization partner for EcR during the vitellogenic response to 20E in the mosquito.

AHR38, a homolog of NGFI-B, inhibits formation of the functional ecdysteroid receptor in the mosquito Aedes aegypti.

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of numerous devastating human diseases. After ingestion of blood, 20-hydroxyecdysone activates yolk protein precursor (YPP) genes in the metabolic tissue, the fat body. An important adaptation for anautogenicity is the previtellogenic developmental arrest (the state-of-arrest) preventing the activation of YPP genes in previtellogenic females prior to blood feeding. Here, we show that a retinoid X receptor homolog, Ultraspiracle (AaUSP), which is an obligatory partner in the functional ecdysteroid receptor, exists at the state-of-arrest as a heterodimer with the orphan nuclear receptor AHR38, a homolog of Drosophila DHR38 and nerve growth factor-induced protein B. Yeast two-hybrid and glutathione S-transferase pull-down assays demonstrate that AHR38 can interact strongly with AaUSP. AHR38 also disrupts binding of ecdysteroid receptor to ecdysone response elements. Cell co-transfection of AHR38 with AaEcR and AaUSP inhibits ecdysone-dependent activation of a reporter gene by the ecdysteroid receptor. Co-immunoprecipitation experiments indicate that AaUSP protein associates with AHR38 instead of AaEcR in fat body nuclei at the state-of-arrest.

Two distinct subpopulations of ecdysone receptor complex in the female mosquito during vitellogenesis.

The native functional ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP) proteins, was identified in the fat body of adult female mosquitoes, Aedes aegypti, through electrophoretic mobility shift assays (EMSA) using previously characterized Drosophila ecdysone response elements (EcREs). The use of different salt concentrations during preparation of nuclear extracts enabled us to characterize two distinct subpopulations of the receptor complex, one of which was high salt-sensitive and responsive to exogenous 20-hydroxyecdysone (20E), and the other of which was high salt-resistant and refractory to exogenous 20E. Salt-sensitivity correlated with ligand responsiveness. Developmental EMSA analyses demonstrated that previtellogenic fat body nuclei and nuclei from the termination phase of vitellogenesis with low 20E titer contained solely high-salt-sensitive, ligand responsive complexes, which could be recovered in nuclear extracts (NEs) only by low salt tissue homogenization, suggesting these complexes were unliganded. In contrast, the fat body nuclei from stages of active vitellogenesis with high 20E titer contained almost exclusively high salt-resistant, ligand refractory complexes, implying these complexes were liganded; the nuclei from the intermediate stages, early and late phases of vitellogenesis, contained a mixture of the two subpopulations. The developmental profile of fully activated, ligand refractory receptor complexes closely correlated with that of yolk protein expression, suggesting an intimate involvement of the ecdysone receptor complex in both the induction and maintenance of high level expression of yolk protein genes.