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Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity
Insect Biochemistry and Molecular Biology 32 (2002) 1275–1286 www.elsevier.com/locate/ibmb
Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity Alexander S. Raikhel a,∗, Vladimir A. Kokoza a, Jinsong Zhu a, David Martin b, ShengFu Wang c, Chao Li d, Guoqiang Sun a, Abdoulaziz Ahmed a, Neal Dittmer e, Geoff Attardo a a
b
Department of Entomology, University of California, Riverside, CA 92521-0314, USA Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CID, CSIC, Barcelona, Spain c Department of Human Genetics, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA d Department of Molecular & Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA e Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA Accepted 15 April 2002
Abstract Elucidation of molecular mechanisms underlying stage- and tissue-specific expression of genes activated by a blood meal is of great importance for current efforts directed towards utilizing molecular genetics to develop novel strategies of mosquito and pathogen control. Regulatory regions of such genes can be used to express anti-pathogen effector molecules in engineered vectors in a precise temporal and spatial manner, designed to maximally affect a pathogen. The fat body is a particularly important target for engineering anti-pathogen properties because in insects, it is a potent secretory tissue releasing its products to the hemolymph, an environment or a crossroad for most pathogens. Recently, we have provided proof of this concept by engineering stable transformant lines of Aedes aegypti mosquito, in which the regulatory region A. aegypti vitellogenin (Vg) gene activates high-level fat body-specific expression of a potent anti-bacterial factor, defensin, in response to a blood meal. Further study of the Vg gene utilizing Drosophila and Aedes transformation identified cis-regulatory sites responsible for state- and fat body-specific activation of this gene via a blood-meal-triggered cascade. 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 a stage-specific hormonal enhancement of the Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic to the Vg gene. Furthermore, our study showed that several transcription factors involved in controlling the Vg gene expression, are themselves targets of the blood meal-mediated regulatory cascade, thus greatly amplifying the effect of this cascade on the Vg gene. This research serves as the foundation for the future design of mosquitospecific expression cassettes with predicted stage- and tissue specificity at the desired levels of transgene expression. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vitellogenin; Ecdysteroid receptor; Transcription factor; Gene regulation; Anautogeny
1. Introduction The yellow fever mosquito, Aedes aegypti, in addition to its importance as a pathogen vector, represents an outstanding model system for arthropod vector research due to the exceptional knowledge base amassed on its physi-
∗
Corresponding author. Tel.: +1-909-787-2129; fax: 909-787-2130. E-mail address: [email protected] (A.S. Raikhel).
ology, biochemistry and development (Clements, 1992). The great wealth of information accumulated over the past two decades concerning vitellogenesis in A. aegypti has made it one of the best studied among insects (reviewed in Raikhel, 1992; Dhadialla and Raikhel, 1994). Hormonal control of mosquito vitellogenesis has been extensively studied and reviewed (Hagedorn, 1985, 1989; Bownes, 1986; Raikhel, 1992; Dhadialla and Raikhel, 1994; Raikhel et al., 1999). The vitellogenic cycle of A. aegypti can be divided into three distinct periods.
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First, in the previtellogenic period, the mosquito undergoes a preparatory phase in which the fat body becomes capable of intense synthesis of yolk protein precursors (YPP). This process is thought to be under the control of juvenile hormone III (JH III). It is followed by a state of arrest which is maintained until a blood meal is taken, after which the mosquito enters the synthetic phase of the vitellogenic period. The fat body then produces YPPs to be accumulated and stored in the yolk bodies of the oocytes. 20-Hydroxyecdysone (20E) is involved in the control of the vitellogenic period (Hagedorn, 1985, 1989; Raikhel, 1992; Dhadialla and Raikhel, 1994; Raikhel et al., 1999). Finally, in the termination period, YPP production is halted in the fat body (Fig. 1). The gene encoding vitellogenin (Vg), a major YPP in most oviparous animals, is expressed in extraovarian tissues in a sex-, tissue- and stage-specific manner. The cDNA encoding A. aegypti Vg has been characterized (Chen et al., 1994), and its gene cloned (Romans et al., 1995). In vitellogenic female insects, the fat body, a powerful metabolic and secretory organ, is engaged in massive production of YPPs for developing oocytes (Raikhel and Dhadialla, 1992). Our studies of the fat body’s role in vitellogenesis in the female mosquito, A. aegypti have shown that it is not limited to the production of Vg. We have discovered that together with Vg, the vitellogenic fat body of Aedes females produces two other YPPs which are pro-enzymes deposited in
developing oocytes and activated during embryogenesis: 53-kDa vitellogenic carboxypeptidase (VCP), and a 44kDa cathepsin B-like protease (VCB). Moreover, the regulation of these YP precursors appears to be similar: they are synthesized exclusively by the fat body in response to a blood meal and maximally expressed at 24 h postblood meal (PBM; Hays and Raikhel, 1990; Cho et al., 1991, 1999). Recently, we have shown that lipophorin (Lp), the insect lipid transport molecule, also plays a role of YPP in A. aegypti (Sun et al., 2000). In anautogenous mosquitoes such as A. aegypti, a unique feature of genes encoding YPPs is the requirement of a blood meal for their activation (Raikhel, 1992). Following blood meal activation, the Vg gene is transcribed at a very high level. The stringent control of the expression of the Vg gene in mosquitoes by the blood meal provides an outstanding model for elucidating hormonal and tissue-specific regulation in the context of complex physiological events surrounding reproduction. Furthermore, the Vg gene is the major regulatory target of the blood meal-activated cascade in the mosquito. Therefore, detailed analysis of the mechanisms governing the expression of this gene is essential. Here, we review recent progress in the understanding of blood meal-activated transcriptional control of Vg and other YPP genes in the mosquito A. aegypti.
Fig. 1. Transcript profiles of major genes of the ecdysteroid regulatory hierarchy during the first cycle of egg maturation in the mosquito, Aedes aegypti. The previtellogenic period begins at eclosion (E) of the adult female. During first 3 days of post-eclosion life of the female, both the fat body and the ovary become competent for subsequent vitellogenesis. The female then enters a state-of-arrest; yolk protein precursors are not synthesized during previtellogenic period. Only when the female mosquito injests blood (BM), vitellogenesis is initiated. Hormones: hormonal titers of juvenile hormone (JH) and ecdysteroids (20E) in A. aegypti females. Fat Body: relative levels of RNAs for nuclear receptors (NRs) determined by RT–PCR. AaEcRA, ecdysone receptor isoform A; AaEcRB, ecdysone receptor isoform B; AaUSPA, Ultraspiracle isoform A; AaUSPB, Ultraspiracle isoform B; AaE74B, E74 isoform B; AaE74A, E74 isoform A; AaE75A, E75 isoform A; AaHR38, mosquito homologue of Drosophila HR38, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP). Modified from Raikhel et al., 1999 (with permission).
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2. Molecular endocrinology of vitellogenesis The hemolymph titers of ecdysteroids in female mosquitoes are correlated with the rate of YPP synthesis in the fat body (Fig. 1). The ecdysteroid titers are only slightly elevated at 4 h PBM; however they rise sharply at 6–8 h PBM, and reach their maximum level at 18–20 h PBM. Numerous studies have clearly established that the ecdysteroid control of vitellogenesis is a central event in the blood meal-activated regulatory cascade leading to successful egg maturation (reviewed in Dhadialla and Raikhel, 1994; Raikhel et al., 1999). In A. aegypti, a preparatory, previtellogenic, developmental period is required for the mosquito fat body to attain competence for 20E responsiveness as well as for the adult female to attain competence for blood feeding. bFTZ-F1, the orphan nuclear factor implicated as a competence factor for stage-specific response to ecdysteroid during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. The transcript of Drosophila bFTZ-F1 homolog is expressed highly in the mosquito fat body during pre- and post-vitellogenic periods when ecdysteroid titers are low. However, there is a delay in appearance of active AaFTZ-F1 factor that coincides with the onset of competence for 20E response (Fig. 2) (Li et al., 2000). In addition, a homolog of Drosophila HR3 is expressed in the vitellogenic tissues of the female mosquito (Kapitskaya et al., 2000). The expression of the mosquito homolog, named AHR3, correlates with the titer of 20E, peaking at late pupa and adult vitellogenic female 24 h PBM, and preceding AaFTZ-F1 expression peaks. The orphan nuclear receptor DHR3, an early–late gene with induction characteristics including properties of both early and late genes, is one of the key genes in the ecdysone-mediated genetic regulatory network. Recent studies have revealed that during insect metamorphosis DHR3 has a dual role in repressing the early genes while activating bFTZ-F1
Fig. 2. βFTZ-F1, the orphan nuclear factor is implicated as a competence factor for stage- specific response to ecdysteroid during in the mosquito fat body. The AaFTZ-F1 mRNA is present at late pupal and previtellogenic stages of newly eclosed female, however the appearance of active AaFTZ-F1 factor coincides with the onset of competence for 20E response (based on Li et al., 2000).
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(White et al., 1997). Thus, the regulation and function of FTZ-F1 during mosquito vitellogenesis closely resembles that shown at the onset of Drosophila metamorphosis and that FTZ-F1 is therefore part of a conserved and broadly utilized molecular mechanism controlling the stage specificity of ecdysteroid response. Analysis of ecdysone effects on polytene chromosome puffing patterns in the late larval and prepupal salivary gland of Drosophila have suggested that the initial activation of a small number of early ecdysone-inducible genes leads to subsequent induction of a large number of late target genes. Elucidation of this genetic hierarchy at the molecular level has led to the identification of the ecdysone receptor as a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor. Furthermore, these studies have shown that the action of the ecdysone–receptor complex is indeed mediated by early genes such as the BR-C, E74 and E75 genes encoding transcription factors involved in regulation of late gene expression. The ecdysone-mediated regulatory network is further refined by the presence of genes that are involved in setting up the timing and stage-specificity of gene activation by this genetic hierarchy (Thummel, 1996). Consistent with the proposed role of 20E in activating mosquito vitellogenesis, experiments using an in vitro fat body culture have shown that physiological doses of 20E (10⫺7–10⫺6 M) activate two YPP genes, Vg and VCP (Deitsch et al., 1995). Transcripts of two different isoforms of EcR, EcR-A and EcR-B, are present in preand vitellogenic ovaries and fat bodies (Wang et al., in press). Transcripts encoding two different USP isoforms are differentially expressed in these tissues as well (Wang et al., 2000) (Fig. 1). The mosquito EcR–USP heterodimer has been shown to bind to various ecdysteroid response elements (EcREs) to modulate ecdysteroid regulation of target genes (Wang et al., 1998). EcREs are present in Vg and VCP genes. Several genes of the ecdysteroid-regulatory hierarchy are conserved between vitellogenesis in mosquitoes and metamorphosis in Drosophila. The A. aegypti homolog of the Drosophila, E75 gene, a representative of the next level in the ecdysteroid response hierarchy, is expressed in the ovary and fat body following a blood meal. Similar to Drosophila, there are three E75 isoforms in A. aegypti that are inducible by 20E. Interestingly, in the mosquito fat body, E75 transcripts show two peaks, with a small peak coinciding with the first peak of 20E. The correlation between mid-vitellogenic expression of the E75 and vitellogenin (Vg) genes suggests that the YPP genes are direct targets of E75 (Pierceall et al., 1999) (Fig. 1). Indeed, analysis of the Vg gene regulatory regions has identified an E75 binding site within a region required for high level Vg expression (Kokoza et al., 2001a). These findings suggest that AaE75 mediates fat
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body ecdysteroid responses, and that the ecdysteroidtriggered regulatory hierarchies, such as those implicated in the initiation of metamorphosis, are reiteratively utilized in the control of the reproductive ecdysteroid response. Two isoforms of the homolog to the Drosophila transcription factor E74, which share a common C-terminal Ets DNA-binding domain, yet have unique N-terminal sequences, are present in the mosquito A. aegypti. They exhibit a high level of identity to DmE74 isoforms A and B and show structural features typical for members of the Ets transcriptional factor superfamily. Furthermore, both mosquito E74 isoforms bind to a Drosophila E74 binding site with the consensus motif C/AGGAA. The AaE74B transcript is induced by a blood meal-activated hormonal cascade in fat bodies and peaks at 24 h PBM, the peak of vitellogenesis (Fig. 1). In contrast, AaE74A is activated at the termination of vitellogenesis, exhibiting a peak at 36 h PBM in the fat body and 48 h PBM in the ovary. These findings suggest that AaE74A and AaE74B isoforms play different roles in regulation of vitellogenesis in mosquitoes, as an activator and a repressor of YPP gene expression respectively (Sun et al., 2002). The Vg gene regulatory portion containing an E74 binding site is required for the high level of Vg expression (Kokoza et al., 2001a). Analysis of the 5⬘-upstream regulatory region of the mosquito Vg gene has revealed the presence of putative binding sites for EcR–USP along with those to the early genes, E74 and E75. This suggests a complex system of regulation of this gene through a combination of direct and indirect hierarchies. The Vg gene contains a functionally active EcRE that binds the heterodimer EcR– USP. A direct repeat with a 1-bp spacer (DR-1) with the sequence AGGCCAaTGGTCG is the major part of the EcRE in the Vg gene (Martin et al., 2001a). Thus, the A. aegypti Vg gene is the target of a direct and indirect regulation by 20E (Fig. 3).
3. Structure and function of regulatory regions of yolk protein genes Transcriptional activation of hormonally controlled, tissue-specific genes involves interactions of sequencespecific transcription factors with enhancer/promoter elements of these genes. It appears that transcription factors of hormonal, stage-specific, and tissue-specific gene regulatory pathways act synergistically to activate transcription of target genes. Our studies have shown synergistic involvement of a number of regulatory elements in governing expression of the Aedes Vg gene. Furthermore, the 5⬘-regulatory region of the gene consists of at least three modules required for its blood-meal activation and high-level expression (Kokoza et al., 2001a). Drosophila and Aedes transformation, as well as DNA-binding
Fig. 3. Direct and indirect regulation of yolk protein precursor genes, Vg by 20-hydroxyecdysone in the mosquito fat body. After binding 20E, the EcR–USP heterodimer activates early genes, E74 and E75, as well as directly acts on the Vg gene allowing its expression. In turn, the products of E74 and E75 genes act as powerful activators of Vg gene transcription (based on Kokoza et al., 2001b; Martin et al., 2001a.
assays, have been used to identify cis-regulatory sites in the Vg gene regulatory region, responsible for stage- and fat body-specific activation of this gene via a bloodmeal-triggered cascade. These analyses revealed three regulatory regions in the 2.1-kb upstream portion of the Vg gene that is sufficient to bring about the characteristic pattern of Vg gene expression (Fig. 4). The proximal region, adjacent to the basal transcription start site, contains binding sites to several transcription factors: EcR/USP, GATA (GATA transcription factor), C/EBP (CAAT-binding protein) and HNF3/fkh (hepatocyte nuclear factor 3/ forkhead transcription factor). This region is required for the correct tissue- and stage-specific expression. It appears that a combinatorial action of these transcription factors is essential to bring a fat body-specific expression. EcR/USP acts as a timer allowing the gene to be turned on. However, the level of expression driven by this regulatory region is low. Analysis of the mosquito Vg gene has revealed that the Vg 5⬘-regulatory region contains a functional ecdysteroid-responsive element (VgEcRE) that is necessary to confer responsiveness to 20E. VgEcRE is directly bound by EcR–USP produced in vitro or from extracts of vitellogenic fat body nuclei. The binding intensity of the EcR–USP–EcRE complex from nuclear extracts corre-
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Fig. 4. Schematic illustration of the regulatory regions of the A. aegypti Vg gene. Numbers refer to nucleotide positions relative to the transcription start site. C/EBP—response element of C/EBP transcription factor; EcRE—ecdysteroid response element; E74 and E75—reponse elements for respective early gene product of the ecdysone hierarchy; GATA—reponse element for GATA trasncription factor; HNF3/fkh—response element for HNF3/forkhead factor; V g—coding region of the Vg gene (from Kokoza et al., 2001a, with permission).
sponds to the levels of ecdysteroids and of the Vg transcript during the vitellogenic cycle. However, 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 (Martin et al., 2001a). The median region carries the sites for early gene factors E74 and E75. It is responsible for a stage-specific hormonal enhancement of the Vg expression. The addition of this region increases expression of the gene in the hormonally controlled manner. Finally, the distal region of the 2.1-kb upstream portion of the Vg gene is characterized by multiple response elements for a transcription factor called GATA. In transgenic experiments utilizing both Aedes and Drosophila, this GATA-rich region has been found to be required for extremely high expression levels characteristic to the Vg gene (Fig. 4). In the mosquito, VCP is the second most abundant YPP. The VCP gene is expressed in synchrony with the Vg gene: both their transcripts appear within 1 h PBM, reach maximal levels at 24 h PBM and rapidly decline to the background levels by 36 h PMB (Fig. 1). 20-Hydroxyecdysone activates both these genes in fat body organ culture and in in vitro cell transfection assay. Furthermore, the same concentration of 20E (10-6M) is required for maximal activation of both these genes. In both genes most of the putative regulatory elements are located in the 2-kb upstream region of the transcription initiation site. Comparative analyses have revealed conservation of putative binding sites for transcription factors responsible for tissue- and stage- specific expression in putative regulatory regions of both genes. Both genes contained sites for the ecdysone receptor (EcRE) and for the early ecdysone-regulatory gene E74. There are also similar tissue-specific binding sites including GATA, C/EBP and HNF3/fkh (Martin, D., Kokoza, V and Raikhel, A.S. unpublished observation). Another level in regulation of YPP gene expression in the mosquito fat body is provided by the control of
transcription factors themselves. Levels of transcripts and active factors critical for transcription of YPP genes such as EcR, USP, E74, E75, and GATA are greatly enhanced during the vitellogenic cycle, and are themselves targets of the blood meal-mediated regulatory cascade (Miura et al., 1999; Pierceall et al., 1999; Kokoza et al., 2001a; Sun et al., 2002). This additional control provides yet another mechanism for amplification of YPP gene expression levels (Figs. 1 and 5).
4. The molecular nature of anautogeny Anautogeny is a fundamental phenomenon underlying the vectorial capacity of mosquitoes. It involves numerous adaptations, from host-seeking behavior to a complete repression of egg maturation prior to a blood meal. The latter is poorly understood at the biochemical and molecular biological levels. Molecular studies of mosquito vitellogenesis have made it possible to elucidate two key mechanisms involved in the YPP gene repression in the fat body of the previtellogenic female mosquito. An important adaptation for anautogeny is the establishment of previtellogenic developmental arrest (the state of arrest) preventing the activation of YPP genes in previtellogenic competent females prior to blood feeding. When an isolated fat body dissected from the previtellogenic female mosquito is incubated in vitro in the presence of physiological doses of 20E, the YPP genes are activated. In contrast, only the injection of 20E at supra-physiological dosages could stimulate some expression of these genes in vivo. Therefore, an inhibition of the 20E-signaling pathway is the essential part of the state of arrest, which may be maintained in vivo by undetermined factors. The disappearance of these factors or appearance of additional, unidentified factors secreted in response to a blood meal may play a crucial role in the release of vitellogenic tissues from the state of arrest.
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can efficiently displace AHR38 and form an active heterodimer with USP, as happens after a blood meal (Fig. 6). A second key element of the repression system is represented by a unique mosquito GATA transcription factor (AaGATAr) that recognizes GATA binding motifs in the upstream region of the YPP genes, Vg and VCP (Martin et al., 2001b) Analyses of tissue and temporal distribution of the AaGATAr transcript have demonstrated that it is expressed only in the fat body of adult female mosquitoes. Furthermore, the active form of AaGATAr exists in nuclei of the fat body at the state of arrest, as well as during postvitellogenic stages when YPP genes are shut off. AaGATAr acts as a repressor of YPP genes by occupying GATA binding sites in regulatory regions of YPP genes. AaGATAr not only inhibits basal YPP gene activity, but it can also overcome ecdy-
Fig. 5. Developmental profile of the native AaEcR–AaUSP ecdysone receptor (A) and AaGATA complexes (B) in A. aegypti fat body nuclear extracts during vitellogenesis. Fat body nuclear extracts were prepared throughout previtellogenesis (PV) and at different hours during the vitellogenic period and subjected to EMSA with 32P-labelled VgEcRE and VgGATA binding sites respectively. (C) Corresponding profiles of the 20E titer (Hagedorn et al., 1975) and the expression of the Vg gene in the fat body (Cho and Raikhel, 1992). BM: blood meal (from Kokoza et al., 2001b, with permission).
In A. aegypti, both AaEcR and AaUSP proteins are abundant in nuclei of the previtellogenic female fat body at the state of arrest; however, the EcR–USP heterodimer capable of binding to the specific EcREs is barely detectable in these nuclei. Studies have shown that the ecdysteroid receptor is a primary target of the 20E signaling modulation in mosquito target tissues at the state of arrest. A possible mechanism through which the formation of ecdysteroid receptor activity can be regulated is a competitive binding of other factors to either EcR or USP. Indeed, at this stage, AaUSP exists as a heterodimer with the orphan nuclear receptor AHR38 (Zhu et al., 2000). AHR38, the mosquito homolog of Drosophila DHR38 and vertebrate NGFI-B/Nurr1 orphan receptors, is a repressor that disrupts the specific DNA binding of the ecdysteroid receptor and interacts strongly with AaUSP. However, in the presence of 10⫺6 M 20E, EcR
Fig. 6. Schematic representation of the mechanism the ecdysteroid receptor is a primary target of the 20E signaling modulation in mosquito target tissues at the state of arrest. At the previtellogenic state of arrest, AaUSP exists as a heterodimer with the repressor AHR38 that prevents formation of the functional ecdysteroid receptor. After a blood meal, EcR displaces AHR38 and forms a functional heterodimer with USP capable of activating ecdysteroid-regulated genes (based on Zhu et al., 2000).
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sone-dependent activation. Impairing ecdysone-dependent activation by AaGATAr during previtellogenesis is essential because at that period, detectable amounts of ecdysteroids as well as the components of the ecdysone receptor, AaEcR and AaUSP, are present. Thus, a member of the GATA-type transcription factor family acts as a tissue-specific repressor of YPP genes in the fat body of the A. aegypti female at the state of arrest. It has been observed that within 0.5 h after a blood meal, the GATA binding sites of the Vg gene are relieved from the AaGATAr binding, and another GATA factor, presumably an activator, binds these sites (Fig. 7). This event coincides with the start of the Vg gene transcription. Future studies should further elucidate this intriguing event in derepression and activation of YPP genes.
5. Engineering blood meal-activated systemic immunity in the mosquito vector Recent progress in mosquito transformation has made it possible to test the idea of engineering anti-pathogen refractoriness, whereby the transmission of a pathogen is impaired by utilizing the transgenic manipulation of its mosquito vector. In such an approach, a chimeric gene consisting of a coding region for a factor with an antipathogen activity driven by a tissue- and stage-specific promoter would be transformed into the vector. Activation of the transgene would result in the production of the anti-pathogen factor that could adversely affect mosquito stages of parasite development, and consequently block its transmission to the vertebrate host. To express potential anti-parasitic factors, we tested the powerful Vg promoter that is activated by a blood meal and highly expressed exclusively in the female mosquito fat body, the center of systemic immunity in insects. We employed the Hermes transposable element as a vector and the Drosophila cinnabar gene as a marker to transform the white-eye A. aegypti host strain. We utilized a 2.1-kb 5⬘upstream region of the Vg gene to drive expression of the defensin A (defA) gene. We tested the regulatory region of the Vg gene of A. aegypti for its ability to express potential anti-pathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg promoter fragment driving the expression of the DefA coding region, one of the major insect immune factors (Fig. 8). PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation (G9), showed that the Vg–DefA transgene insertion was stable. The Vg–DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24 h PBM, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin (tDefA) were accumulated in the hemolymph of blood-fed female mosquitoes, persisting
Fig. 7. Schematic representation of the events of repression and derepression of the Vg gene during the state of arrest and blood meal dependent activation via GATA transcription factors. At the state of arrest, GATA binding motifs in the upstream region of the Vg gene are occupied by a unique GATA repressor transcription factor that prevents the gene from being activated. The unknown factors released as a result of blood feeding results in replacement of GATA repressor by GATA activator and derepression of the gene (based on Martin et al., 2001b).
for 20–22 days after a single blood feeding (Fig. 9). Purified tDefA showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes (Fig. 10). Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood-meal-triggered cascade rather than by infection. This work represents a significant step towards the development of novel molecular genetic approaches to the control of vector competence in pathogen transmission. The development of fat body-specific promoter construct, activated by blood meal triggered cascade,
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Fig. 8. (A) Eye color phenotypes of the A. aegypti Wild-type Rockefeller/UGAL strain, the red eye pH[cn][Vg-DefA] transgenic strain and white eye khW strain. (B) A schematic diagram of the pH[cn][Vg-DefA] construct.
will allow the testing of numerous effector molecules being developed in other laboratories for their antipathogen properties. In summary, a 2.1-kb Vg upstream promoter fragment was sufficient for activation of the transgene by a blood meal-triggered regulatory cascade in a tissue-specific manner. It was also adequate to drive a high level of the transgene expression. Active defensin peptide is produced by the transgene and persists for as long as 22 days PBM. The major significance of our present work is demonstrating that it is conceptually possible to engineer a transgenic mosquito in which turning on the gene(s) encoding anti-pathogen factors is “wired” to the blood meal activation cascade. Thus, it constitutes a crucial step towards the utilization of molecular transgenesis in the development of pathogen-refractory vectors. Further progress largely depends on developing a highly efficient routine gene transformation for vector insects. We tested the piggyBac transposable vector pBac[3×P3-EGFP afm] for transformation of A. aegypti. Previously, the selectable marker, EGFP, under the 3×P3 promoter has been shown to be expressed in the eyes of two different insects, Drosophila melanogaster and the flour beetle Tribolium castaneum (Berghammer et al., 1999). We constructed a vector containing pBac[3×P3EGFP afm] and the Vg–def transgene and transformed the A. aegypti white-eye khw mutant strain. We generated several stable transformant lines that showed intense
fluorescence in their eyes (Fig. 11A). In two independent transgenic lines so far established, the EGFP expression pattern was uniform and level of expression in the eyes was very high at all developmental stages, starting from the first instar larvae. Molecular analysis demonstrated that integration events occurred through a canonical cutand-paste mechanism resulted in perfect incorporation of pBac[3×P3-EGFP afm]–Vg–def construct into the mosquito genome. The stability and integrity of transgenes were confirmed from G1 to G5 generations, and genetic data demonstrated Mendelian segregation. Importantly, the strong fluorescence was detected at larval and pupal stages even after several subsequent outcrossings (from G1 to G5) to wild-type Rockefeller/Ugal strain (Fig. 11B). These results clearly indicate that the 3×P3-EGFP marker can be used for microinjection directly into wild type Aedes and Anopheles eggs. In addition, the Vg–Def transgene was strongly activated in the fat body after blood meal and its expression demonstrated stage-, tissue- and sex-specificity in all transgenic strains with white-eye and wild-type genetic backgrounds (Kokoza et al., 2001b).
6. Conclusions and future directions Mosquitoes transmit many diseases which are among the most threatening in modern times. The major reasons
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Fig. 9. Immunoblot analyses of tDefA peptide expression in transgenic mosquitoes after blood meal activation. (A) Protein extracts from individual 24 h PBM females of the host khW strain (khW) and transgenic D1 strain (D1) were tested using polyclonal antibody to A. aegypti defensin A and monoclonal antibody to A. aegypti vitellogenin small subunit (27). Expression of the 4-kDa defensin A peptide was observed in transgenic mosquitoes but not in the khW host strain. The expression of 66-kDa Vg-small subunit (Vg-S), used as a positive control for blood meal activation, was present at the same level in both the transgenic strain and host strain. (B) Immunoblot analysis of tissueand sex-specific expression of tDefA peptide in transgenic mosquitoes. Protein extracts from hemolymph (H), fat bodies (FB), ovary (OV), carcass (C), 24 h PBM whole mosquito female, (F), mosquito male (M) were analyzed. (C) Time course of tDefA protein accumulation in transgenic mosquitoes during the vitellogenic cycle after a single blood feeding. Previtellogenic female (PV), vitellogenic females 1 (1d), 3(3d), 22 (22d) days PBM and the hemolymph collected from 22-day-old female mosquitoes (H22d) were tested using defensin Aspecific antibodies. One mosquito-equivalent was loaded in each lane, except for the hemolymph sample from 22-day-old females, where a four mosquito-equivalent was used (from Kokoza et al., 2000).
for this catastrophic situation are unavailability of effective anti-malarial vaccines, other mosquito-borne diseases and the development of insecticide and drug resistance by the vectors and pathogens, respectively. Therefore, there is an urgent need to explore every possible avenue for developing novel control strategies against these menacing mosquito-borne diseases.
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Fig. 10. Analysis of antibacterial activity of tDefA peptide isolated from transgenic mosquitoes. (A) Immunoblot analysis of peptide fractions used to assay the antibacterial activity. Protein samples obtained from transgenic 24 h PBM females of D1 strain (TR), bacterially induced non-transgenic females of the khW host strain (BI), and nontransgenic 24 h PBM females of the khW host strain (NT), were isolated using acid extraction and chromatography purification on Sep-Pak C18 cartridges and analyzed by immunoblotting. The analyzed samples were prepared from 0% acetonitrile fraction in acidified water (0%) as a loading control, and 40% acetonitrile fraction (40%), enriched for defensin A peptide. (B) Aliquots from 40% fractions containing the major portion of defensin, isolated from transgenic (TR) and bacteriainduced (BI) females, were analyzed by the liquid growth inhibition assay using the Gram-positive bacteria, Micrococcus luteus. An equal aliquot of the 40% acetonitrile fraction isolated from non-transgenic 24 h PBM females of the host strain (NT) was used as a negative control. Data represent the mean of three independent experiments (from Kokoza et al., 2000).
The female mosquito acquires pathogens with a blood meal that also activates numerous genes essential for digestion of the blood, synthesis of yolk protein precursors and ultimately, production of eggs. Thus, elucidation of the molecular mechanisms underlying tissueand stage-specific YPP gene expression is of great importance to the current efforts directed toward utilizing molecular genetics to develop novel strategies in vector control, Significant progress has been achieved in the biochemical and molecular characterization of key
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Fig. 11. Expression of the 3×P3-EGFP selectable marker in the eyes of transgenic A. aegypti. (A) EGFP expression in adult eyes of transgenic khw mosquitoes (white-eye strain) transformed by pBac[3×P3-EGFP]Vg–DefA. (B) Expression and visibility of the EGFP selectable marker in the eyes of a black eye hybrid third instar larva, resulted from the crossing of a khw transgenic female with a wild-type (Rockefeller/UGAL) untransformed male. The visibility of the EGFP expression in the mosquitoes with black-pigmented eyes was limited to the larval and pupal stages. The left panels are fluorescent images, while the right panels bright-light images. (C) Schematic representation of the PiggyBac[3×P3-EGFP]Vg–DefA construct (from Kokoza et al., 2001b, with permission).
genes expressed in the fat body of the vector model, the mosquito, A. aegypti, during vitellogenesis. This progress provides the framework for further studies of molecular pathways leading to expression of fat body-specific genes, as well as of the molecular basis for anautogenicity in other vectors.
The powerful promoters of blood meal-activated fat body-specific genes are among the best candidates for construction of chimeric genes incorporating immune factors or other physiologically important molecules. These chimeric genes would be highly expressed in the fat body in response to a blood meal, and their products
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secreted to the hemolymph, an ideal milieu for combating a pathogen. A transgenic mosquito with the blood meal activated systemic immunity has been engineered by utilizing the Vg promoter and coding region of defensin gene (Def). In this transgenic mosquito, the Def is activated by a blood meal, and the fat body produces and secretes large amounts of biologically active defensin. This work has provided proof of the concept that anti-pathogen effector molecules can be stably transformed into vectors and be expressed in a precisely timed fashion engineered to maximally effect a pathogen (Fig. 12). In addition, it has clearly demonstrated the importance of fundamental research on the key physiological events triggered by a blood meal. Anautogeny is the most fundamental phenomena underlying the strict control of vitellogenesis by intake of blood or protein meal. As a consequence, anautogeny is the sole reason that hematophagous arthropods are vectors of numerous pathogens. Our studies have begun to elucidate the biochemical and molecular biological mechanisms. Two molecular mechanisms have been elucidated in the mosquito A. aegvpti: sequestration of USP and binding of a GATA repressor protein, as discussed above. More studies should be done to further elucidate molecular and genetic details of these mechanisms. It will be also important to understand how universal these mechanisms are among other groups of arthropod vectors, particularly those distantly related to mosquitoes. In conclusion, the elucidation of the molecular mechanisms underlying tissue- and stage-specific gene expression in the mosquito fat body is of great importance to the current efforts directed toward utilizing molecular genetics to develop novel control strategies. Significant progress has been achieved in the biochemical and molecular characterization of key genes expressed
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in the fat body of the model mosquito, A. aegypti, during vitellogenesis. This progress provides the framework for further studies of molecular pathways leading to expression of fat body-specific genes, as well as basis for anautogeny in mosquito vectors. Acknowledgements The authors are grateful to Dr David Arnosti for critical reading and MS Megan Ackroyd for editing the manuscript. This work was supported by grants from the National Institutes of Health. References Berghammer, A.J., Klinger, M., Wimmer, E.A., 1999. A universal marker for transgenic insects. Nature 402, 370–371. Bownes, M., 1986. Expression of the genes coding for vitellogenin (yolk protein). Ann. Rev. Entomol. 31, 507–531. Chen, J.-S., Cho, W.L., Raikhel, A.S., 1994. Mosquito vitellogenin cDNA. Sequence similarity with vertebrate vitellogenin and insect larval hemolymph proteins. J. Molec. Biol. 237, 641–647. Cho, W.L., Raikhel, A.S., 1992. Cloning of cDNA for Mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E. J. Biol. Chem. 267, 21823–21829. Cho, W.L., Deitsch, K.W., Raikhel, A.S., 1991. An extraovarian protein accumulated in mosquito oocytes in a carboxypeptidase activated in embryos. Proc. Natl. Acad. Sci. USA 88, 10821–10824. Cho, W.L., Tsao, S.M., Hays, A.R., Walter, R., Chen, J.S., Snigirevskaya, E.S., Raikhel, A.S., 1999. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J. Biol. Chem. 274, 13311–13321. Clements, A.N., 1992. The Biology of Mosquitoes vol. 1. Development, Nutrition, and Reproduction. Chapman & Hall, New York. Deitsch, K.W., Chen, J.S., Raikhel, A.S., 1995. Indirect control of yolk protein genes by 20-hydroxyecdysone in the fat body of the mosquito, Aedes aegypti. Insect Biochem. Molec. Biol. 25, 449–454.
Fig. 12. Schematic representation of the engineered blood meal-induced fat body-specific genetic drive.
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Dhadialla, T.S., Raikhel, A.S., 1994. Endocrinology of mosquito vitellogenesis. In: Perspectives in Comparative Endocrinology. National Research Council of Canada, Canada, pp. 275–281. Hagedorn, H.H., 1985. The role of ecdysteroids in reproduction. In: Kerkut, G., Gilbert, L. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 8. Pergamon Press, Oxford, UK, pp. 205–261. Hagedorn, H.H., 1989. Physiological roles of hemolymph ecdysteroid in the adult insect. In: Koolman, J. (Ed.), Ecdysone from Chemistry to Mode of Action. New York, Thieme Medical Publishers, Inc, pp. 279–289. Hays, A.R., Raikhel, A.S., 1990. A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes. Roux’s Archives of Developmental Biology 199, 114–121. Kapitskaya, M.Z., Li, C., Miura, K., Segroves, W., Raikhel, A.S., 2000. 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. Mol. Cell. Endocrinol 160, 25–37. Kokoza, V.A., Ahmed, A., Cho, W.-L., Jasinskiene, N., James, A., Raikhel, A.S., 2000. Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. USA 97, 1624–1629. Kokoza, V.A., Martin, D., Mienaltowski, M., Ahmed, A., Morton, C., Raikhel, A.S., 2001a. Transcriptional regulation of the mosquito Aedes aegypti vitellogenin gene by a blood-meal triggered cascade. Gene 274, 47–65. Kokoza, V.A., Ahmed, A.S., Wimmer, E., Raikhel, A.S., 2001b. Efficient transformation of the yellow fever mosquito Aedes aegypti using the pBac[3xP3-EGFP afm] piggy-Bac transposable element vector. Insect Biochem. Molec. Biol. 31, 1137–1143. Li, C., Kapitskaya, M.Z., Zhu, J.S., Miura, K., Segraves, W.A., Raikhel, A.S., 2000. Conserved mechanism for stage specificity of the mosquito vitellogenic response to ecdysone. Develop. Biol. 224, 96–110. Martin, D., Wang, S.-F., Raikhel, A.S., 2001a. The vitellogenin gene of the mosquito Aedes aegypti is a direct target of the ecdysteroid receptor. Molec. Cell. Endocrinol. 173, 75–86. Martin, D., Piulachs, M.D., Raikhel, A.S., 2001b. A novel GATA fac-
tor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with CtBP corepressor. Molec. Cell. Biol. 21, 164–174. Pierceall, W.E., Li, C., Biran, A., Miura, K., Raikhel, A.S., Segraves, W.A., 1999. E75 expression in the mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction. Molec.Cell. Endocrinol. 150, 73–89. Raikhel, A.S., 1992. Vitellogenesis in mosquitoes. Adv. Dis. Vector Res. 9, 1–39. Raikhel, A.S., Dhadialla, T.S., 1992. Accumulation of yolk proteins in insect oocytes. Annu. Rev. Entomol. 37, 217–251. Raikhel, A.S., Miura, K., Segraves, W.A., 1999. Nuclear receptors in mosquito vitellogenesis. Amer. Zoologist 39, 722–735. Romans, P., Tu, Z., Ke, Z., Hagedorn, H.H., 1995. Analysis of a vitellogenin gene of the mosquito, Aedes aegypti and comparisons to vitellogenins from other organisms. Insect Biochem. Molec. Biol. 25, 939–958. Sun, G., Zhu, J.S., Li, C.L., Tu, J., Raikhel, A.S., 2002. Two isoforms of the early E74 gene, an Ets transcription factor homologue, are utilized in the ecdysteriod hierarchy governing vitellogenesis of the mosquito Aedes asgypti. Molec. Cell. Endocrinol 190, 147–157. Sun, J., Hiraoka, T., Dittmer, N.T., Cho, K.H., Raikhel, A.S., 2000. Lipophorin as a yolk protein in the mosquito, Aedes aegypti. Insect Biochem. Molec. Biol. 30, 1161–1171. Thummel, C.S., 1996. Flies on steroids—Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends Genetics 12, 306–310. Wang, S., Miura, K., Miksicek, R.J., Segraves, W.A., Raikhel, A.S., 1998. DNA-binding properties of mosquito ecdysteroid receptor/Ultraspiracle heterodimers. J. Biol. Chem. 273, 27531– 27540. Wang, S.F., Li, C., Zhu, J.S., Raikhel, A.S. Differential expression and regulation of mosquito ecdysteriod receptor isoforms by 20hydroxyecdysone. Molec. Cell. Endocrinol. (in press). White, K.P., Hurban, P., Watanabe, T., Hogness, D.S., 1997. Coordination of Drosophilia metamorphosis by two ecdysone-induced nuclear receptors. Science 276, 114–117. Zhu, J.S., Miura, K., Chen, L., Raikhel, A.S., 2000. AHR38, a homolog of NGFI-B, inhibits formation of the functional ecdysteroid receptor in the mosquito Aedes aegypti. EMBO J. 19, 253–262.
Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity Alexander S. Raikhel a,∗, Vladimir A. Kokoza a, Jinsong Zhu a, David Martin b, ShengFu Wang c, Chao Li d, Guoqiang Sun a, Abdoulaziz Ahmed a, Neal Dittmer e, Geoff Attardo a a
b
Department of Entomology, University of California, Riverside, CA 92521-0314, USA Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CID, CSIC, Barcelona, Spain c Department of Human Genetics, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA d Department of Molecular & Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA e Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA Accepted 15 April 2002
Abstract Elucidation of molecular mechanisms underlying stage- and tissue-specific expression of genes activated by a blood meal is of great importance for current efforts directed towards utilizing molecular genetics to develop novel strategies of mosquito and pathogen control. Regulatory regions of such genes can be used to express anti-pathogen effector molecules in engineered vectors in a precise temporal and spatial manner, designed to maximally affect a pathogen. The fat body is a particularly important target for engineering anti-pathogen properties because in insects, it is a potent secretory tissue releasing its products to the hemolymph, an environment or a crossroad for most pathogens. Recently, we have provided proof of this concept by engineering stable transformant lines of Aedes aegypti mosquito, in which the regulatory region A. aegypti vitellogenin (Vg) gene activates high-level fat body-specific expression of a potent anti-bacterial factor, defensin, in response to a blood meal. Further study of the Vg gene utilizing Drosophila and Aedes transformation identified cis-regulatory sites responsible for state- and fat body-specific activation of this gene via a blood-meal-triggered cascade. 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 a stage-specific hormonal enhancement of the Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic to the Vg gene. Furthermore, our study showed that several transcription factors involved in controlling the Vg gene expression, are themselves targets of the blood meal-mediated regulatory cascade, thus greatly amplifying the effect of this cascade on the Vg gene. This research serves as the foundation for the future design of mosquitospecific expression cassettes with predicted stage- and tissue specificity at the desired levels of transgene expression. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vitellogenin; Ecdysteroid receptor; Transcription factor; Gene regulation; Anautogeny
1. Introduction The yellow fever mosquito, Aedes aegypti, in addition to its importance as a pathogen vector, represents an outstanding model system for arthropod vector research due to the exceptional knowledge base amassed on its physi-
∗
Corresponding author. Tel.: +1-909-787-2129; fax: 909-787-2130. E-mail address: [email protected] (A.S. Raikhel).
ology, biochemistry and development (Clements, 1992). The great wealth of information accumulated over the past two decades concerning vitellogenesis in A. aegypti has made it one of the best studied among insects (reviewed in Raikhel, 1992; Dhadialla and Raikhel, 1994). Hormonal control of mosquito vitellogenesis has been extensively studied and reviewed (Hagedorn, 1985, 1989; Bownes, 1986; Raikhel, 1992; Dhadialla and Raikhel, 1994; Raikhel et al., 1999). The vitellogenic cycle of A. aegypti can be divided into three distinct periods.
0965-1748/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 2 ) 0 0 0 9 0 - 5
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First, in the previtellogenic period, the mosquito undergoes a preparatory phase in which the fat body becomes capable of intense synthesis of yolk protein precursors (YPP). This process is thought to be under the control of juvenile hormone III (JH III). It is followed by a state of arrest which is maintained until a blood meal is taken, after which the mosquito enters the synthetic phase of the vitellogenic period. The fat body then produces YPPs to be accumulated and stored in the yolk bodies of the oocytes. 20-Hydroxyecdysone (20E) is involved in the control of the vitellogenic period (Hagedorn, 1985, 1989; Raikhel, 1992; Dhadialla and Raikhel, 1994; Raikhel et al., 1999). Finally, in the termination period, YPP production is halted in the fat body (Fig. 1). The gene encoding vitellogenin (Vg), a major YPP in most oviparous animals, is expressed in extraovarian tissues in a sex-, tissue- and stage-specific manner. The cDNA encoding A. aegypti Vg has been characterized (Chen et al., 1994), and its gene cloned (Romans et al., 1995). In vitellogenic female insects, the fat body, a powerful metabolic and secretory organ, is engaged in massive production of YPPs for developing oocytes (Raikhel and Dhadialla, 1992). Our studies of the fat body’s role in vitellogenesis in the female mosquito, A. aegypti have shown that it is not limited to the production of Vg. We have discovered that together with Vg, the vitellogenic fat body of Aedes females produces two other YPPs which are pro-enzymes deposited in
developing oocytes and activated during embryogenesis: 53-kDa vitellogenic carboxypeptidase (VCP), and a 44kDa cathepsin B-like protease (VCB). Moreover, the regulation of these YP precursors appears to be similar: they are synthesized exclusively by the fat body in response to a blood meal and maximally expressed at 24 h postblood meal (PBM; Hays and Raikhel, 1990; Cho et al., 1991, 1999). Recently, we have shown that lipophorin (Lp), the insect lipid transport molecule, also plays a role of YPP in A. aegypti (Sun et al., 2000). In anautogenous mosquitoes such as A. aegypti, a unique feature of genes encoding YPPs is the requirement of a blood meal for their activation (Raikhel, 1992). Following blood meal activation, the Vg gene is transcribed at a very high level. The stringent control of the expression of the Vg gene in mosquitoes by the blood meal provides an outstanding model for elucidating hormonal and tissue-specific regulation in the context of complex physiological events surrounding reproduction. Furthermore, the Vg gene is the major regulatory target of the blood meal-activated cascade in the mosquito. Therefore, detailed analysis of the mechanisms governing the expression of this gene is essential. Here, we review recent progress in the understanding of blood meal-activated transcriptional control of Vg and other YPP genes in the mosquito A. aegypti.
Fig. 1. Transcript profiles of major genes of the ecdysteroid regulatory hierarchy during the first cycle of egg maturation in the mosquito, Aedes aegypti. The previtellogenic period begins at eclosion (E) of the adult female. During first 3 days of post-eclosion life of the female, both the fat body and the ovary become competent for subsequent vitellogenesis. The female then enters a state-of-arrest; yolk protein precursors are not synthesized during previtellogenic period. Only when the female mosquito injests blood (BM), vitellogenesis is initiated. Hormones: hormonal titers of juvenile hormone (JH) and ecdysteroids (20E) in A. aegypti females. Fat Body: relative levels of RNAs for nuclear receptors (NRs) determined by RT–PCR. AaEcRA, ecdysone receptor isoform A; AaEcRB, ecdysone receptor isoform B; AaUSPA, Ultraspiracle isoform A; AaUSPB, Ultraspiracle isoform B; AaE74B, E74 isoform B; AaE74A, E74 isoform A; AaE75A, E75 isoform A; AaHR38, mosquito homologue of Drosophila HR38, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP). Modified from Raikhel et al., 1999 (with permission).
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2. Molecular endocrinology of vitellogenesis The hemolymph titers of ecdysteroids in female mosquitoes are correlated with the rate of YPP synthesis in the fat body (Fig. 1). The ecdysteroid titers are only slightly elevated at 4 h PBM; however they rise sharply at 6–8 h PBM, and reach their maximum level at 18–20 h PBM. Numerous studies have clearly established that the ecdysteroid control of vitellogenesis is a central event in the blood meal-activated regulatory cascade leading to successful egg maturation (reviewed in Dhadialla and Raikhel, 1994; Raikhel et al., 1999). In A. aegypti, a preparatory, previtellogenic, developmental period is required for the mosquito fat body to attain competence for 20E responsiveness as well as for the adult female to attain competence for blood feeding. bFTZ-F1, the orphan nuclear factor implicated as a competence factor for stage-specific response to ecdysteroid during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. The transcript of Drosophila bFTZ-F1 homolog is expressed highly in the mosquito fat body during pre- and post-vitellogenic periods when ecdysteroid titers are low. However, there is a delay in appearance of active AaFTZ-F1 factor that coincides with the onset of competence for 20E response (Fig. 2) (Li et al., 2000). In addition, a homolog of Drosophila HR3 is expressed in the vitellogenic tissues of the female mosquito (Kapitskaya et al., 2000). The expression of the mosquito homolog, named AHR3, correlates with the titer of 20E, peaking at late pupa and adult vitellogenic female 24 h PBM, and preceding AaFTZ-F1 expression peaks. The orphan nuclear receptor DHR3, an early–late gene with induction characteristics including properties of both early and late genes, is one of the key genes in the ecdysone-mediated genetic regulatory network. Recent studies have revealed that during insect metamorphosis DHR3 has a dual role in repressing the early genes while activating bFTZ-F1
Fig. 2. βFTZ-F1, the orphan nuclear factor is implicated as a competence factor for stage- specific response to ecdysteroid during in the mosquito fat body. The AaFTZ-F1 mRNA is present at late pupal and previtellogenic stages of newly eclosed female, however the appearance of active AaFTZ-F1 factor coincides with the onset of competence for 20E response (based on Li et al., 2000).
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(White et al., 1997). Thus, the regulation and function of FTZ-F1 during mosquito vitellogenesis closely resembles that shown at the onset of Drosophila metamorphosis and that FTZ-F1 is therefore part of a conserved and broadly utilized molecular mechanism controlling the stage specificity of ecdysteroid response. Analysis of ecdysone effects on polytene chromosome puffing patterns in the late larval and prepupal salivary gland of Drosophila have suggested that the initial activation of a small number of early ecdysone-inducible genes leads to subsequent induction of a large number of late target genes. Elucidation of this genetic hierarchy at the molecular level has led to the identification of the ecdysone receptor as a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), the insect homolog of the vertebrate retinoid X receptor. Furthermore, these studies have shown that the action of the ecdysone–receptor complex is indeed mediated by early genes such as the BR-C, E74 and E75 genes encoding transcription factors involved in regulation of late gene expression. The ecdysone-mediated regulatory network is further refined by the presence of genes that are involved in setting up the timing and stage-specificity of gene activation by this genetic hierarchy (Thummel, 1996). Consistent with the proposed role of 20E in activating mosquito vitellogenesis, experiments using an in vitro fat body culture have shown that physiological doses of 20E (10⫺7–10⫺6 M) activate two YPP genes, Vg and VCP (Deitsch et al., 1995). Transcripts of two different isoforms of EcR, EcR-A and EcR-B, are present in preand vitellogenic ovaries and fat bodies (Wang et al., in press). Transcripts encoding two different USP isoforms are differentially expressed in these tissues as well (Wang et al., 2000) (Fig. 1). The mosquito EcR–USP heterodimer has been shown to bind to various ecdysteroid response elements (EcREs) to modulate ecdysteroid regulation of target genes (Wang et al., 1998). EcREs are present in Vg and VCP genes. Several genes of the ecdysteroid-regulatory hierarchy are conserved between vitellogenesis in mosquitoes and metamorphosis in Drosophila. The A. aegypti homolog of the Drosophila, E75 gene, a representative of the next level in the ecdysteroid response hierarchy, is expressed in the ovary and fat body following a blood meal. Similar to Drosophila, there are three E75 isoforms in A. aegypti that are inducible by 20E. Interestingly, in the mosquito fat body, E75 transcripts show two peaks, with a small peak coinciding with the first peak of 20E. The correlation between mid-vitellogenic expression of the E75 and vitellogenin (Vg) genes suggests that the YPP genes are direct targets of E75 (Pierceall et al., 1999) (Fig. 1). Indeed, analysis of the Vg gene regulatory regions has identified an E75 binding site within a region required for high level Vg expression (Kokoza et al., 2001a). These findings suggest that AaE75 mediates fat
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body ecdysteroid responses, and that the ecdysteroidtriggered regulatory hierarchies, such as those implicated in the initiation of metamorphosis, are reiteratively utilized in the control of the reproductive ecdysteroid response. Two isoforms of the homolog to the Drosophila transcription factor E74, which share a common C-terminal Ets DNA-binding domain, yet have unique N-terminal sequences, are present in the mosquito A. aegypti. They exhibit a high level of identity to DmE74 isoforms A and B and show structural features typical for members of the Ets transcriptional factor superfamily. Furthermore, both mosquito E74 isoforms bind to a Drosophila E74 binding site with the consensus motif C/AGGAA. The AaE74B transcript is induced by a blood meal-activated hormonal cascade in fat bodies and peaks at 24 h PBM, the peak of vitellogenesis (Fig. 1). In contrast, AaE74A is activated at the termination of vitellogenesis, exhibiting a peak at 36 h PBM in the fat body and 48 h PBM in the ovary. These findings suggest that AaE74A and AaE74B isoforms play different roles in regulation of vitellogenesis in mosquitoes, as an activator and a repressor of YPP gene expression respectively (Sun et al., 2002). The Vg gene regulatory portion containing an E74 binding site is required for the high level of Vg expression (Kokoza et al., 2001a). Analysis of the 5⬘-upstream regulatory region of the mosquito Vg gene has revealed the presence of putative binding sites for EcR–USP along with those to the early genes, E74 and E75. This suggests a complex system of regulation of this gene through a combination of direct and indirect hierarchies. The Vg gene contains a functionally active EcRE that binds the heterodimer EcR– USP. A direct repeat with a 1-bp spacer (DR-1) with the sequence AGGCCAaTGGTCG is the major part of the EcRE in the Vg gene (Martin et al., 2001a). Thus, the A. aegypti Vg gene is the target of a direct and indirect regulation by 20E (Fig. 3).
3. Structure and function of regulatory regions of yolk protein genes Transcriptional activation of hormonally controlled, tissue-specific genes involves interactions of sequencespecific transcription factors with enhancer/promoter elements of these genes. It appears that transcription factors of hormonal, stage-specific, and tissue-specific gene regulatory pathways act synergistically to activate transcription of target genes. Our studies have shown synergistic involvement of a number of regulatory elements in governing expression of the Aedes Vg gene. Furthermore, the 5⬘-regulatory region of the gene consists of at least three modules required for its blood-meal activation and high-level expression (Kokoza et al., 2001a). Drosophila and Aedes transformation, as well as DNA-binding
Fig. 3. Direct and indirect regulation of yolk protein precursor genes, Vg by 20-hydroxyecdysone in the mosquito fat body. After binding 20E, the EcR–USP heterodimer activates early genes, E74 and E75, as well as directly acts on the Vg gene allowing its expression. In turn, the products of E74 and E75 genes act as powerful activators of Vg gene transcription (based on Kokoza et al., 2001b; Martin et al., 2001a.
assays, have been used to identify cis-regulatory sites in the Vg gene regulatory region, responsible for stage- and fat body-specific activation of this gene via a bloodmeal-triggered cascade. These analyses revealed three regulatory regions in the 2.1-kb upstream portion of the Vg gene that is sufficient to bring about the characteristic pattern of Vg gene expression (Fig. 4). The proximal region, adjacent to the basal transcription start site, contains binding sites to several transcription factors: EcR/USP, GATA (GATA transcription factor), C/EBP (CAAT-binding protein) and HNF3/fkh (hepatocyte nuclear factor 3/ forkhead transcription factor). This region is required for the correct tissue- and stage-specific expression. It appears that a combinatorial action of these transcription factors is essential to bring a fat body-specific expression. EcR/USP acts as a timer allowing the gene to be turned on. However, the level of expression driven by this regulatory region is low. Analysis of the mosquito Vg gene has revealed that the Vg 5⬘-regulatory region contains a functional ecdysteroid-responsive element (VgEcRE) that is necessary to confer responsiveness to 20E. VgEcRE is directly bound by EcR–USP produced in vitro or from extracts of vitellogenic fat body nuclei. The binding intensity of the EcR–USP–EcRE complex from nuclear extracts corre-
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Fig. 4. Schematic illustration of the regulatory regions of the A. aegypti Vg gene. Numbers refer to nucleotide positions relative to the transcription start site. C/EBP—response element of C/EBP transcription factor; EcRE—ecdysteroid response element; E74 and E75—reponse elements for respective early gene product of the ecdysone hierarchy; GATA—reponse element for GATA trasncription factor; HNF3/fkh—response element for HNF3/forkhead factor; V g—coding region of the Vg gene (from Kokoza et al., 2001a, with permission).
sponds to the levels of ecdysteroids and of the Vg transcript during the vitellogenic cycle. However, 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 (Martin et al., 2001a). The median region carries the sites for early gene factors E74 and E75. It is responsible for a stage-specific hormonal enhancement of the Vg expression. The addition of this region increases expression of the gene in the hormonally controlled manner. Finally, the distal region of the 2.1-kb upstream portion of the Vg gene is characterized by multiple response elements for a transcription factor called GATA. In transgenic experiments utilizing both Aedes and Drosophila, this GATA-rich region has been found to be required for extremely high expression levels characteristic to the Vg gene (Fig. 4). In the mosquito, VCP is the second most abundant YPP. The VCP gene is expressed in synchrony with the Vg gene: both their transcripts appear within 1 h PBM, reach maximal levels at 24 h PBM and rapidly decline to the background levels by 36 h PMB (Fig. 1). 20-Hydroxyecdysone activates both these genes in fat body organ culture and in in vitro cell transfection assay. Furthermore, the same concentration of 20E (10-6M) is required for maximal activation of both these genes. In both genes most of the putative regulatory elements are located in the 2-kb upstream region of the transcription initiation site. Comparative analyses have revealed conservation of putative binding sites for transcription factors responsible for tissue- and stage- specific expression in putative regulatory regions of both genes. Both genes contained sites for the ecdysone receptor (EcRE) and for the early ecdysone-regulatory gene E74. There are also similar tissue-specific binding sites including GATA, C/EBP and HNF3/fkh (Martin, D., Kokoza, V and Raikhel, A.S. unpublished observation). Another level in regulation of YPP gene expression in the mosquito fat body is provided by the control of
transcription factors themselves. Levels of transcripts and active factors critical for transcription of YPP genes such as EcR, USP, E74, E75, and GATA are greatly enhanced during the vitellogenic cycle, and are themselves targets of the blood meal-mediated regulatory cascade (Miura et al., 1999; Pierceall et al., 1999; Kokoza et al., 2001a; Sun et al., 2002). This additional control provides yet another mechanism for amplification of YPP gene expression levels (Figs. 1 and 5).
4. The molecular nature of anautogeny Anautogeny is a fundamental phenomenon underlying the vectorial capacity of mosquitoes. It involves numerous adaptations, from host-seeking behavior to a complete repression of egg maturation prior to a blood meal. The latter is poorly understood at the biochemical and molecular biological levels. Molecular studies of mosquito vitellogenesis have made it possible to elucidate two key mechanisms involved in the YPP gene repression in the fat body of the previtellogenic female mosquito. An important adaptation for anautogeny is the establishment of previtellogenic developmental arrest (the state of arrest) preventing the activation of YPP genes in previtellogenic competent females prior to blood feeding. When an isolated fat body dissected from the previtellogenic female mosquito is incubated in vitro in the presence of physiological doses of 20E, the YPP genes are activated. In contrast, only the injection of 20E at supra-physiological dosages could stimulate some expression of these genes in vivo. Therefore, an inhibition of the 20E-signaling pathway is the essential part of the state of arrest, which may be maintained in vivo by undetermined factors. The disappearance of these factors or appearance of additional, unidentified factors secreted in response to a blood meal may play a crucial role in the release of vitellogenic tissues from the state of arrest.
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can efficiently displace AHR38 and form an active heterodimer with USP, as happens after a blood meal (Fig. 6). A second key element of the repression system is represented by a unique mosquito GATA transcription factor (AaGATAr) that recognizes GATA binding motifs in the upstream region of the YPP genes, Vg and VCP (Martin et al., 2001b) Analyses of tissue and temporal distribution of the AaGATAr transcript have demonstrated that it is expressed only in the fat body of adult female mosquitoes. Furthermore, the active form of AaGATAr exists in nuclei of the fat body at the state of arrest, as well as during postvitellogenic stages when YPP genes are shut off. AaGATAr acts as a repressor of YPP genes by occupying GATA binding sites in regulatory regions of YPP genes. AaGATAr not only inhibits basal YPP gene activity, but it can also overcome ecdy-
Fig. 5. Developmental profile of the native AaEcR–AaUSP ecdysone receptor (A) and AaGATA complexes (B) in A. aegypti fat body nuclear extracts during vitellogenesis. Fat body nuclear extracts were prepared throughout previtellogenesis (PV) and at different hours during the vitellogenic period and subjected to EMSA with 32P-labelled VgEcRE and VgGATA binding sites respectively. (C) Corresponding profiles of the 20E titer (Hagedorn et al., 1975) and the expression of the Vg gene in the fat body (Cho and Raikhel, 1992). BM: blood meal (from Kokoza et al., 2001b, with permission).
In A. aegypti, both AaEcR and AaUSP proteins are abundant in nuclei of the previtellogenic female fat body at the state of arrest; however, the EcR–USP heterodimer capable of binding to the specific EcREs is barely detectable in these nuclei. Studies have shown that the ecdysteroid receptor is a primary target of the 20E signaling modulation in mosquito target tissues at the state of arrest. A possible mechanism through which the formation of ecdysteroid receptor activity can be regulated is a competitive binding of other factors to either EcR or USP. Indeed, at this stage, AaUSP exists as a heterodimer with the orphan nuclear receptor AHR38 (Zhu et al., 2000). AHR38, the mosquito homolog of Drosophila DHR38 and vertebrate NGFI-B/Nurr1 orphan receptors, is a repressor that disrupts the specific DNA binding of the ecdysteroid receptor and interacts strongly with AaUSP. However, in the presence of 10⫺6 M 20E, EcR
Fig. 6. Schematic representation of the mechanism the ecdysteroid receptor is a primary target of the 20E signaling modulation in mosquito target tissues at the state of arrest. At the previtellogenic state of arrest, AaUSP exists as a heterodimer with the repressor AHR38 that prevents formation of the functional ecdysteroid receptor. After a blood meal, EcR displaces AHR38 and forms a functional heterodimer with USP capable of activating ecdysteroid-regulated genes (based on Zhu et al., 2000).
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sone-dependent activation. Impairing ecdysone-dependent activation by AaGATAr during previtellogenesis is essential because at that period, detectable amounts of ecdysteroids as well as the components of the ecdysone receptor, AaEcR and AaUSP, are present. Thus, a member of the GATA-type transcription factor family acts as a tissue-specific repressor of YPP genes in the fat body of the A. aegypti female at the state of arrest. It has been observed that within 0.5 h after a blood meal, the GATA binding sites of the Vg gene are relieved from the AaGATAr binding, and another GATA factor, presumably an activator, binds these sites (Fig. 7). This event coincides with the start of the Vg gene transcription. Future studies should further elucidate this intriguing event in derepression and activation of YPP genes.
5. Engineering blood meal-activated systemic immunity in the mosquito vector Recent progress in mosquito transformation has made it possible to test the idea of engineering anti-pathogen refractoriness, whereby the transmission of a pathogen is impaired by utilizing the transgenic manipulation of its mosquito vector. In such an approach, a chimeric gene consisting of a coding region for a factor with an antipathogen activity driven by a tissue- and stage-specific promoter would be transformed into the vector. Activation of the transgene would result in the production of the anti-pathogen factor that could adversely affect mosquito stages of parasite development, and consequently block its transmission to the vertebrate host. To express potential anti-parasitic factors, we tested the powerful Vg promoter that is activated by a blood meal and highly expressed exclusively in the female mosquito fat body, the center of systemic immunity in insects. We employed the Hermes transposable element as a vector and the Drosophila cinnabar gene as a marker to transform the white-eye A. aegypti host strain. We utilized a 2.1-kb 5⬘upstream region of the Vg gene to drive expression of the defensin A (defA) gene. We tested the regulatory region of the Vg gene of A. aegypti for its ability to express potential anti-pathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg promoter fragment driving the expression of the DefA coding region, one of the major insect immune factors (Fig. 8). PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation (G9), showed that the Vg–DefA transgene insertion was stable. The Vg–DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24 h PBM, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin (tDefA) were accumulated in the hemolymph of blood-fed female mosquitoes, persisting
Fig. 7. Schematic representation of the events of repression and derepression of the Vg gene during the state of arrest and blood meal dependent activation via GATA transcription factors. At the state of arrest, GATA binding motifs in the upstream region of the Vg gene are occupied by a unique GATA repressor transcription factor that prevents the gene from being activated. The unknown factors released as a result of blood feeding results in replacement of GATA repressor by GATA activator and derepression of the gene (based on Martin et al., 2001b).
for 20–22 days after a single blood feeding (Fig. 9). Purified tDefA showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes (Fig. 10). Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood-meal-triggered cascade rather than by infection. This work represents a significant step towards the development of novel molecular genetic approaches to the control of vector competence in pathogen transmission. The development of fat body-specific promoter construct, activated by blood meal triggered cascade,
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Fig. 8. (A) Eye color phenotypes of the A. aegypti Wild-type Rockefeller/UGAL strain, the red eye pH[cn][Vg-DefA] transgenic strain and white eye khW strain. (B) A schematic diagram of the pH[cn][Vg-DefA] construct.
will allow the testing of numerous effector molecules being developed in other laboratories for their antipathogen properties. In summary, a 2.1-kb Vg upstream promoter fragment was sufficient for activation of the transgene by a blood meal-triggered regulatory cascade in a tissue-specific manner. It was also adequate to drive a high level of the transgene expression. Active defensin peptide is produced by the transgene and persists for as long as 22 days PBM. The major significance of our present work is demonstrating that it is conceptually possible to engineer a transgenic mosquito in which turning on the gene(s) encoding anti-pathogen factors is “wired” to the blood meal activation cascade. Thus, it constitutes a crucial step towards the utilization of molecular transgenesis in the development of pathogen-refractory vectors. Further progress largely depends on developing a highly efficient routine gene transformation for vector insects. We tested the piggyBac transposable vector pBac[3×P3-EGFP afm] for transformation of A. aegypti. Previously, the selectable marker, EGFP, under the 3×P3 promoter has been shown to be expressed in the eyes of two different insects, Drosophila melanogaster and the flour beetle Tribolium castaneum (Berghammer et al., 1999). We constructed a vector containing pBac[3×P3EGFP afm] and the Vg–def transgene and transformed the A. aegypti white-eye khw mutant strain. We generated several stable transformant lines that showed intense
fluorescence in their eyes (Fig. 11A). In two independent transgenic lines so far established, the EGFP expression pattern was uniform and level of expression in the eyes was very high at all developmental stages, starting from the first instar larvae. Molecular analysis demonstrated that integration events occurred through a canonical cutand-paste mechanism resulted in perfect incorporation of pBac[3×P3-EGFP afm]–Vg–def construct into the mosquito genome. The stability and integrity of transgenes were confirmed from G1 to G5 generations, and genetic data demonstrated Mendelian segregation. Importantly, the strong fluorescence was detected at larval and pupal stages even after several subsequent outcrossings (from G1 to G5) to wild-type Rockefeller/Ugal strain (Fig. 11B). These results clearly indicate that the 3×P3-EGFP marker can be used for microinjection directly into wild type Aedes and Anopheles eggs. In addition, the Vg–Def transgene was strongly activated in the fat body after blood meal and its expression demonstrated stage-, tissue- and sex-specificity in all transgenic strains with white-eye and wild-type genetic backgrounds (Kokoza et al., 2001b).
6. Conclusions and future directions Mosquitoes transmit many diseases which are among the most threatening in modern times. The major reasons
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Fig. 9. Immunoblot analyses of tDefA peptide expression in transgenic mosquitoes after blood meal activation. (A) Protein extracts from individual 24 h PBM females of the host khW strain (khW) and transgenic D1 strain (D1) were tested using polyclonal antibody to A. aegypti defensin A and monoclonal antibody to A. aegypti vitellogenin small subunit (27). Expression of the 4-kDa defensin A peptide was observed in transgenic mosquitoes but not in the khW host strain. The expression of 66-kDa Vg-small subunit (Vg-S), used as a positive control for blood meal activation, was present at the same level in both the transgenic strain and host strain. (B) Immunoblot analysis of tissueand sex-specific expression of tDefA peptide in transgenic mosquitoes. Protein extracts from hemolymph (H), fat bodies (FB), ovary (OV), carcass (C), 24 h PBM whole mosquito female, (F), mosquito male (M) were analyzed. (C) Time course of tDefA protein accumulation in transgenic mosquitoes during the vitellogenic cycle after a single blood feeding. Previtellogenic female (PV), vitellogenic females 1 (1d), 3(3d), 22 (22d) days PBM and the hemolymph collected from 22-day-old female mosquitoes (H22d) were tested using defensin Aspecific antibodies. One mosquito-equivalent was loaded in each lane, except for the hemolymph sample from 22-day-old females, where a four mosquito-equivalent was used (from Kokoza et al., 2000).
for this catastrophic situation are unavailability of effective anti-malarial vaccines, other mosquito-borne diseases and the development of insecticide and drug resistance by the vectors and pathogens, respectively. Therefore, there is an urgent need to explore every possible avenue for developing novel control strategies against these menacing mosquito-borne diseases.
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Fig. 10. Analysis of antibacterial activity of tDefA peptide isolated from transgenic mosquitoes. (A) Immunoblot analysis of peptide fractions used to assay the antibacterial activity. Protein samples obtained from transgenic 24 h PBM females of D1 strain (TR), bacterially induced non-transgenic females of the khW host strain (BI), and nontransgenic 24 h PBM females of the khW host strain (NT), were isolated using acid extraction and chromatography purification on Sep-Pak C18 cartridges and analyzed by immunoblotting. The analyzed samples were prepared from 0% acetonitrile fraction in acidified water (0%) as a loading control, and 40% acetonitrile fraction (40%), enriched for defensin A peptide. (B) Aliquots from 40% fractions containing the major portion of defensin, isolated from transgenic (TR) and bacteriainduced (BI) females, were analyzed by the liquid growth inhibition assay using the Gram-positive bacteria, Micrococcus luteus. An equal aliquot of the 40% acetonitrile fraction isolated from non-transgenic 24 h PBM females of the host strain (NT) was used as a negative control. Data represent the mean of three independent experiments (from Kokoza et al., 2000).
The female mosquito acquires pathogens with a blood meal that also activates numerous genes essential for digestion of the blood, synthesis of yolk protein precursors and ultimately, production of eggs. Thus, elucidation of the molecular mechanisms underlying tissueand stage-specific YPP gene expression is of great importance to the current efforts directed toward utilizing molecular genetics to develop novel strategies in vector control, Significant progress has been achieved in the biochemical and molecular characterization of key
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Fig. 11. Expression of the 3×P3-EGFP selectable marker in the eyes of transgenic A. aegypti. (A) EGFP expression in adult eyes of transgenic khw mosquitoes (white-eye strain) transformed by pBac[3×P3-EGFP]Vg–DefA. (B) Expression and visibility of the EGFP selectable marker in the eyes of a black eye hybrid third instar larva, resulted from the crossing of a khw transgenic female with a wild-type (Rockefeller/UGAL) untransformed male. The visibility of the EGFP expression in the mosquitoes with black-pigmented eyes was limited to the larval and pupal stages. The left panels are fluorescent images, while the right panels bright-light images. (C) Schematic representation of the PiggyBac[3×P3-EGFP]Vg–DefA construct (from Kokoza et al., 2001b, with permission).
genes expressed in the fat body of the vector model, the mosquito, A. aegypti, during vitellogenesis. This progress provides the framework for further studies of molecular pathways leading to expression of fat body-specific genes, as well as of the molecular basis for anautogenicity in other vectors.
The powerful promoters of blood meal-activated fat body-specific genes are among the best candidates for construction of chimeric genes incorporating immune factors or other physiologically important molecules. These chimeric genes would be highly expressed in the fat body in response to a blood meal, and their products
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secreted to the hemolymph, an ideal milieu for combating a pathogen. A transgenic mosquito with the blood meal activated systemic immunity has been engineered by utilizing the Vg promoter and coding region of defensin gene (Def). In this transgenic mosquito, the Def is activated by a blood meal, and the fat body produces and secretes large amounts of biologically active defensin. This work has provided proof of the concept that anti-pathogen effector molecules can be stably transformed into vectors and be expressed in a precisely timed fashion engineered to maximally effect a pathogen (Fig. 12). In addition, it has clearly demonstrated the importance of fundamental research on the key physiological events triggered by a blood meal. Anautogeny is the most fundamental phenomena underlying the strict control of vitellogenesis by intake of blood or protein meal. As a consequence, anautogeny is the sole reason that hematophagous arthropods are vectors of numerous pathogens. Our studies have begun to elucidate the biochemical and molecular biological mechanisms. Two molecular mechanisms have been elucidated in the mosquito A. aegvpti: sequestration of USP and binding of a GATA repressor protein, as discussed above. More studies should be done to further elucidate molecular and genetic details of these mechanisms. It will be also important to understand how universal these mechanisms are among other groups of arthropod vectors, particularly those distantly related to mosquitoes. In conclusion, the elucidation of the molecular mechanisms underlying tissue- and stage-specific gene expression in the mosquito fat body is of great importance to the current efforts directed toward utilizing molecular genetics to develop novel control strategies. Significant progress has been achieved in the biochemical and molecular characterization of key genes expressed
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in the fat body of the model mosquito, A. aegypti, during vitellogenesis. This progress provides the framework for further studies of molecular pathways leading to expression of fat body-specific genes, as well as basis for anautogeny in mosquito vectors. Acknowledgements The authors are grateful to Dr David Arnosti for critical reading and MS Megan Ackroyd for editing the manuscript. This work was supported by grants from the National Institutes of Health. References Berghammer, A.J., Klinger, M., Wimmer, E.A., 1999. A universal marker for transgenic insects. Nature 402, 370–371. Bownes, M., 1986. Expression of the genes coding for vitellogenin (yolk protein). Ann. Rev. Entomol. 31, 507–531. Chen, J.-S., Cho, W.L., Raikhel, A.S., 1994. Mosquito vitellogenin cDNA. Sequence similarity with vertebrate vitellogenin and insect larval hemolymph proteins. J. Molec. Biol. 237, 641–647. Cho, W.L., Raikhel, A.S., 1992. Cloning of cDNA for Mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E. J. Biol. Chem. 267, 21823–21829. Cho, W.L., Deitsch, K.W., Raikhel, A.S., 1991. An extraovarian protein accumulated in mosquito oocytes in a carboxypeptidase activated in embryos. Proc. Natl. Acad. Sci. USA 88, 10821–10824. Cho, W.L., Tsao, S.M., Hays, A.R., Walter, R., Chen, J.S., Snigirevskaya, E.S., Raikhel, A.S., 1999. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J. Biol. Chem. 274, 13311–13321. Clements, A.N., 1992. The Biology of Mosquitoes vol. 1. Development, Nutrition, and Reproduction. Chapman & Hall, New York. Deitsch, K.W., Chen, J.S., Raikhel, A.S., 1995. Indirect control of yolk protein genes by 20-hydroxyecdysone in the fat body of the mosquito, Aedes aegypti. Insect Biochem. Molec. Biol. 25, 449–454.
Fig. 12. Schematic representation of the engineered blood meal-induced fat body-specific genetic drive.
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