Nuclear receptors in the mosquito Aedes aegypti Annotation, hormonal regulation and expression profiling Josefa Cruz*, Douglas H Sieglaff*, , Peter Arensburger, Peter W Atkinson and Alexander S Raikhel Department of Entomology and the Institute for Integrative Genome Biology, University of California, Riverside, CA, USA Keywords ecdysone receptor; 20-hydroxyecdysone; nuclear receptor; reproduction; yolk protein Correspondence A S Raikhel, Department of Entomology, University of California, Riverside, Watkins Drive, CA 92521, USA Fax: +1 951 827 2140 Tel: +1 951 827 2146 E-mail: araikhel@ucr.edu  Present address Institute for Genomics and Bioinformatics Microbiology and Molecular Genetics, University of California, Irvine, CA, USA *These authors contributed equally to this work (Received 29 May 2008, revised 11 December 2008, accepted 16 December 2008) doi:10.1111/j.1742-4658.2008.06860.x In anautogenous mosquitoes, egg development requires blood feeding and as a consequence mosquitoes act as vectors of numerous devastating diseases of humans and domestic animals Understanding the molecular mechanisms regulating mosquito egg development may contribute significantly to the development of novel vector-control strategies Previous studies have shown that in the yellow fever mosquito Aedes aegypti, nuclear receptors (NRs) play a key role in the endocrine regulation of reproduction However, many mosquito NRs remain uncharacterized, some of which may play an important role in mosquito reproduction Publication of the genome of A aegypti allowed us to identify all NRs in this mosquito based on their phylogenetic relatedness to those within Insecta We have determined that there are 20 putative A aegypti NRs, some of which are predicted to have different isoforms As the first step toward analysis of this gene family, we have established their expression within the two main reproductive tissues of adult female mosquitoes: fat body and ovary All NR transcripts are present in both tissues, most displaying dynamic expression profiles during reproductive cycles Finally, in vitro assays with isolated fat bodies were conducted to identify the role of the steroid hormone 20-hydroxyecdysone in modulating the expression of A aegypti NRs These data which describe the identification, expression and hormonal regulation of 20 NRs in the yellow fever mosquito lay a solid foundation for future studies on the hormonal regulation of reproduction in mosquitoes Mosquitoes are vectors of some of the world’s most devastating diseases Malaria causes approximately million deaths annually (http://www.who.org) and dengue, a rapidly expanding disease in most tropical and subtropical areas of the world, has become the most significant arboviral disease of humans Anopheline mosquitoes are the vectors of malaria, whereas Aedes species is the vector of dengue and yellow fever Both disease vectors are exquisitely adapted to living around humans and using human blood as a nutrient source to promote egg development A basic understanding of mosquito reproductive biology is an important component in developing novel strategies for use in the control of mosquito-borne disease Egg maturation in Aedes aegypti adult females includes a process termed vitellogenesis, which involves massive production of yolk protein precursors (YPPs) by the fat body and their subsequent internalization into the developing oocyte, helping to support later embryonic development The two primary insect Abbreviations Chx, cycloheximide; 20E, 20-hydroxyecdysone; EcR, ecdysone receptor; JH III, juvenile hormone III; NR, nuclear receptors; PBM, post blood meal; Vg, vitellogenin; YPP, yolk protein precursors FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1233 Nuclear receptors in Aedes aegypti J Cruz et al hormones governing vitellogenesis are the sesquiterpenoid juvenile hormone III (JH III) and the steroid 20hydroxyecdysone (20E) The period following adult eclosion requires JH III to promote the development of ‘competence’ or the ability of the female mosquito to process the blood meal in the promotion of vitellogenesis [1,2]; 72 h is typically required after eclosion to achieve this state This development toward ‘competence’ is termed pre-vitellogenesis JH III titer levels are highest in adult females during pre-vitellogenic development, but fall dramatically after ingestion of the blood meal; the 20E concentration, however, begins to increase within a few hours post blood meal (PBM), peaking at 18–24 h PBM [3] 20E is one of the primary regulators in the synthesis of vitellogenin (Vg), the main YPP protein produced by the fat body [4,5] The molecular mechanism of 20E action has been dissected in detail in studies of Drosophila melanogaster development [6–9] The functional 20E receptor is composed of two proteins, the ecdysone receptor (EcR), which binds specifically to 20E, and the product of the ultraspiracle gene, USP [10,11] Once the EcR ⁄ USP complex binds 20E, the heterodimer elicits the expression of a set of genes, including hormonal receptor (Hr3 or Hr46), HR4, HR39, E75, E78 and fushi tarazu transcription factor (ftz-f1)[12] Subsequently, the products of these genes alone, or in combination with other factors, activate late effector genes that control downstream physiological responses All the aforementioned factors, together with EcR and USP, are members of the nuclear receptor (NR) superfamily A similar 20E regulatory pathway is utilized for the promotion of vitellogenesis in A aegypti Before the female mosquito takes a blood meal, both AaEcR and AaUSP proteins are present in fat body cells; however, the AaEcR ⁄ AaUSP heterodimer is barely detectable Indeed, at this stage, AaUSP is prevented from associating with AaEcR by the orphan NR AaHR38, and only after a blood meal is taken and the 20E titer increases can AaEcR efficiently displace AaHR38 to form the AaEcR ⁄ AaUSP heterodimer [13] It has been shown that the AaEcR ⁄ AaUSP heterodimer directly binds the Vg promoter, thereby activating its expression [14] As stated previously, JH III promotes the acquisition of competence in the fat body during the first days following adult eclosion, and has been shown to coordinate development of competence through its ability to promote the translation of another NR, AabFTZ-F1 [15] Following a blood meal, AabFTZF1 promotes EcR activity by recruiting the coactivator p160 ⁄ SRC (AaFISC) which, in turn, binds the AaEcR ⁄ AaUSP heterodimers, establishing a functional 1234 multiple protein complex on the Vg promoter [16] By 24 h PBM, AaVg transcript levels reach their maximum, after which they sharply decline, concluding with the termination of vitellogenesis In this termination process, mosquito Seven-up (AaSvp), a NR member, plays a central role replacing AaUSP in the AaEcR ⁄ AaUSP heterodimer complex, thereby blocking the action of 20E [13] Another A aegypti NR, AaHNF-4c, has also been proposed to promote the termination of Vg expression [17], but the mechanism is still unknown The regulation of AaVg gene expression by 20E acts not only through members of the NR family, but also through other transcription factors such as E74, Ets-domain protein and Broadcomplex, C2H2-type zinc-finger DNA-binding protein [18,19] Despite the achievements mentioned so far, there are additional NRs that remain uncharacterized in the mosquito, some of which may play an important role in its reproduction In this study, we identified and began to characterize all putative NRs of A aegypti We report the annotation of 19 canonical NR family members along with one member of the so-called Knirps group In addition, we determined the expression profiles for transcripts of these NRs within two reproductive tissues of the adult female mosquito – the fat body and ovaries Furthermore, using an in vitro fat body culture system allowed us to identify NRs responsive to 20E This work provides a foundation from which future studies in post-genomic functional analysis of NR developmental regulation in mosquitoes can begin Results and Discussion Identification of NRs in the genome of A aegypti We identified A aegypti NRs from the 1.0 Genebuild assembly of the A aegypti genome (created from a merge of the TIGR and VectorBase 0.5 annotation sets) Protein homology searches were performed using individual members of the NR family from D melanogaster compared against the A aegypti database (http://aaegypti.vectorbase.org/index.php) We identified 20 NR family members in the A aegypti genome, 19 of which are likely orthologs of D melanogaster NRs, and 1which is a likely ortholog of the Apis mellifera and Tribolium castaneum PNR-like NR [20–22] Following our manual initial annotation, some of our in silico predicted sequences were split into unassembled automatic predicted sequences in the A aegypti genomic database; with some given splice sites and exons not predicted in our original manual annotation FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al Nuclear receptors in Aedes aegypti To address these discrepancies, we conducted RT-PCR and 5¢-RACE analysis against AaHR96, AaPNR-like, AaHR4 and AaHR83 All experimentally confirmed sequences have been deposited in GenBank, and the corresponding accession numbers are provided in Table The competence factor bFTZ-F1 of A aegypti was first cloned by Li et al [23] from a cDNA library prepared from the fat bodies of vitellogenic female mosquitoes They isolated several clones that code for a single protein Interestingly, during the initial process of identifying the NR family members in the A aegypti genome, we predicted two isoforms of AaFTZ-F1, differing only in their A ⁄ B region, as observed for the D melanogaster FTZ-F1 isoforms [24,25] Initially, we hypothesized that the mosquito isoforms would be related to those described for D melanogaster, but with a low percentage of identity in the A ⁄ B domain between the two (data not shown) We designated the isoforms of this A aegypti NR as AabFTZ-F1A (previously named AabFTZ-F1) [23] and AabFTZ-F1B Phylogenetic analysis We conducted a phylogenetic analysis of the NRs from the five insect genomes sequenced so far: D melanogaster, Anopheles gambiae, Ap mellifera, T castaneum and A aegypti as well as the sequences of the human orthologs When different isoforms were recovered, only the longest amino acid sequences that included the DNA binding, hinge and ligand-binding domains were used for this analysis, except for the Knirps family members that lack the LBD In D melanogaster, Table NRs of Aedes aegypti NR family members according to the NuReBASE proposed nomenclature [27] General names are based on the nomenclature of D melanogaster with the exception of PNR-like, which has not been annotated in D melanogaster, and is named according to the Ap mellifera ortholog A aegypti NR name and isoform, if present VectorBase and NCBI Accession numbers The NRs transcriptionally controlled by 20E, in an in vitro fat body culture system are indicated in the 20E response column NT, not tested; P, primary response; S, secondary response; NR, not responsive genes NR family member General name A aegypti name NR0A2 NR1D3 Knirps-like Ecdysone-induced protein 75B AaKnrl AaEip75B NR1E1 AaEip78C NR1F4 NR1H1 Ecdysone-induced protein 78C Hormone receptor-like in 46 Ecdysone receptor NR1J1 NR2A4 Hormone receptor-like in 96 Hepatocyte nuclear factor AaHr96 AaHnf4 NR2B4 Ultraspiracle AaUSP NR2D1 NR2E2 NR2E3 NR2E4 NR2E5 NR2E6 NR2F3 Hormone receptor-like in 78 Tailless Hormone receptor-like in 51 Dissatisfaction Hormone receptor-like in 83 PNR-like Seven up AaHr78 AaTll AaHr51 AaDsf AaHr83 AaPNR-like AaSvp NR3B4 NR4A4 NR5A3 Estrogen-related receptor Hormone receptor-like in 38 Ftz transcription factor AaERR AaHr38 AaFTZ-F1 NR5B1 NR6A1 Hormone receptor-like in 39 Hormone receptor-like in Isoform AaHr39 AaHr4 AaHr46 AaEcR AaE75A AaE75B AaE75C AaEcRA AaEcRB AaHnf4-A AaHnf4-B AaHnf4-C AaUSP-A AaUSP-B AabFTZ-F1A AabFTZ-F1B FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS Vector base accession no NCBI accession no 20E response AAEL011231 AAEL007397 BN001172 AM397060 AM397061 AM397062 AM773442 NT P P P P AF230281 AY345989 AF305214 AM773443 AF059026 AF059027 AF059028 AF305213 AF305214 P P NR NT NR NR S NR S BN001173 BN001174 BN001175 BN001178 AM773444 AM773445 AF303224 P NR NT NT NT NR NR BN001176 AF165528 AF274870 AM773446 BN001177 AM773447 NR NR NR S P P 24839.m08763, AAEL000327 AAEL009588 AAEL010719, AAEL009600 AAEL010715, AAEL009600 AAEL007202, AAEL007214 AAEL011323 AAEL000395 VBTranscript 011769, AAEL000395 AAEL001796 AAEL003020 AAEL007190 AAEL005381 AAEL007350 AAEL008043, AAEL008047 AAEL006916, AAEL002765, AAEL002768 AAEL013546 AAEL013270 AAEL002053, AAEL002062 AAEL002062 AAEL001304 AAEL005850, AAEL005864 1235 Nuclear receptors in Aedes aegypti J Cruz et al this group is composed of three members (knirps, knirps-like and eagle) In A aegypti, we only characterized one member of the Knirps group that presented higher amino acid similarity with Dmknirps-like (31%) than with Dmknirps (23%) or DmEg (25%, data not shown) Remarkably, both A aegypti and An gambiae genomes contain only one Knirps family member (knirps-like, Table S1) As previously mentioned, we identified 19 canonical NR family members in the A aegypti genome, 18 of which are likely orthologs of D melanogaster, An gambiae and Ap mellifera NRs, and one which is a likely ortholog of the Ap mellifera and T castaneum PNR-like NR [20–22] that is also present in the genome of An gambiae (Table S1 and Fig 1) Our phylogenetic analysis supported the clustering of this NR in the subfamily NR2E beside the NRs Hr-83, Hr-51, Tll and Dsf, as previously classified by Velaverde et al and Bonneton et al [20,22], but not with the hypothesis that this NR receptor was lost in the dipteran lineage, as demonstrated by its presence in both mosquito genomes analyzed in this study (Fig and Table S1), as well as in the genome of the mosquito Culex quinquefasciatus (http://www vectorbase.org/, vector base gene id: CPIJ017885, data not shown) Six ancestral NR subfamilies have been defined by means of phylogenetic analyses of vertebrate as well as Caenorhabditis elegans and D melanogaster NRs [26– 29] In our phylogenetic analysis, we clustered the NRs from the five insect genomes according to these six subfamilies (Fig and Table S1), indicating that the classification proposed for the A aegypti NRs is supported by phylogenetic consistency The only discrepancy in our phylogenetic analysis is present in the NR2B group The topology of our tree showed that the vertebrate sequence (HsRXR) clustered with the non-dipteran species sequences (TcUSP and AmUSP), but with low support (53% bootstrap) Diptera had the longest branch length, clearly indicating a much more rapid rate of divergence compared with other insects and vertebrate sequences, as previously reported [30,31] Expression in adult female reproductive tissues To determine whether NR family members are expressed in the two main reproductive tissues of adult female mosquitoes, we conducted an initial assessment using RT-PCR Total RNA was extracted from the fat body and ovaries of pre-vitellogenic females 5–6 days after eclosion and from vitellogenic females 6–12 and 18–24 h after a blood meal, and then was subjected to 1236 RT-PCR with a specific primer pair for each NR (see Table S2 for primer sequences) Two biological replicates were analyzed, and Fig depicts the profile matching both replicates An increase in transcript abundance for AaEcRA, AaEcRB, AaE75A, AaE75B, AaE75C, AaHR3, AaHR4, AaE78 and AaHR39 occurred in both tissues, correlating with the known rise in ecdysteroids in vivo, whereas AaUSP-B, AaHR38, AaTll and AaPNR-like only displayed an increase in transcript abundance in the fat body during this same period (Fig 2) This suggests that these later orphan NRs may be hormone inducible, which we addressed using in vitro assays (see below) Many NR transcripts displayed a decrease during the initial phase of vitellogenesis (6 + 12 h PBM) only to increase again at peak vitellogenesis (18 + 24 h PBM) These transcripts (AaUSP-A, AabFTZ-F1A, AabFTZ-F1B, AaHR78, AaHNF-4A, AaHNF-4B, AaHNF-4C, AaSvp and AaERR) were also analyzed in our in vitro assay Any kind of fluctuation in expression levels in the developmental time point addressed in our study for four additional NRs (AaHR83, AaHR51, AaDsf and AaHR96) was difficult to detect using our methods In the hopes of gaining a better clarification of their developmental expression patterns, we increased the number of PCR cycles for both fat body and ovary samples for many NR transcripts studied Such an effort did not lead to better resolution (not shown) In D melanogaster, DmDsf is expressed in a group of neurons in the central nervous system and is required for normal sexual behavior [32] FAX-1, the ortholog of HR51 and HR83 in C elegans, is required for neurotransmitter expression in specific interneurons [33], and the human ortholog, PNR, displays a highly restricted expression in retinal tissues [34] These studies imply a tissue-restricted expression of the members of this NR family, suggesting that what we observed may have simply been basal level expression, not discernable using our RT-PCR analysis By contrast to this restricted expression observed within nervous systems, HR96 has been implicated in regulating xenobiotic responses in D melanogaster and is highly expressed in tissues that monitor and metabolize xenobiotics, including the fat body [35] It has also been established that HR96 is broadly expressed throughout larval development and metamorphosis [7] Given the expression profiles displayed in Fig 2, we decided not to conduct further qPCR analysis against AaHr83, AaHR51, AaDsf and AaHR96 As observed in Fig 2, the majority of NRs are expressed at a constant level within the ovaries during the period analyzed However, AaE75A, AaE75B, FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al Nuclear receptors in Aedes aegypti Fig Phylogenetic tree of insect NRs The different NR families are organized into groupings, NR1–NR6 The tree was constructed following the distance-based neighbor-joining method, using the NRs sequences of D melanogaster (Dm), A aegypti (Aa), An gambiae (Ag), Ap mellifera (Am) and T castaneum (Tc) indicated in Table S1 as well as the human (H sapiens, Hs) orthologs obtained from Genebank Branch lengths are proportional to sequence divergence The bar represents 0.1 substitutions per site The bootstraps nodal support values are shown FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1237 Nuclear receptors in Aedes aegypti J Cruz et al has been extensively studied in development and metamorphosis in insects, its potential role in promoting oogenesis has received significantly less attention EcR mutant females of D melanogaster display abnormal egg chamber development and loss of vitellogenic egg stages [38] as well as chorion malformations [39] In the cockroach Blattella germanica, females treated with BgEcRA dsRNA displayed a reduction in the number of follicular cells in the basal oocyte, subsequent nymphal developmental defects and even a reorganization of the follicular epithelium in the resulting adults [40] DmE75A and DmE75B have been implicated in defining the transition stages and of the egg chambers through either inducing or suppressing apoptosis of the nurse cells [41,42] However, in B mori, BmE75 mediates the transition from vitellogenesis to choriogenesis [43] Although the presence of dynamic expression profiles of various NR family members within Insecta suggests the reiterative use of ecdysone-regulatory hierarchies in fat body and ovary reproductive functions throughout this class, the complexity of the meroistic ovaries of mosquitoes makes extrapolation of function for these NRs from the condition in other insects very difficult That is, each ovariole in mosquitoes consists of a germline-derived oocyte and nurse cells surrounded by somatically derived follicle cells; with our methodology, we are unable to distinguish among these three distinctive cell types [44] Thus, the NR transcripts present in our ovary samples could either be involved in ovary development or comprise a maternal contribution for later embryonic development Indeed, nine NR transcripts are maternally loaded in D melanogaster ovaries [7] Expression and 20E regulation of A aegypti NRs in the fat body Fig Expression of A aegypti nuclear receptors (NRs) in fat body (FB) and ovary (Ov) of pre-vitellogenic female mosquitoes (PV) 4–5 days after eclosion and at 6–12 or 18–24 h after a blood meal (PBM) was determined by quantitative PCR The profiles are representative of two biological replicates AaE75C and AaHR3 mRNAs increased with the in vivo ecdysteroid peak, corroborating results from previous studies [36,37] Such an increase in expression levels within the ovary along with known ecdysteroid titers in vivo was also observed for AaEcRA, AaEcRB, AaHR4, AaE78, AaHNF-4A and AaHNF4C transcripts AaSvp was the only one that displayed a reduction in mRNA levels in ovaries at 18–24 h PBM (Fig 2) While the ecdysone response hierarchy 1238 In order to determine a more complete profile of A aegypti NR expression within the fat body before, during and after vitellogenesis, we conducted qPCR against those NRs that had displayed dynamic expression within the fat body in our earlier RT-PCR experiment Total RNA was isolated from the fat body of three independent collections of mosquito females staged at different time points during pre-vitellogenic and vitellogenic stages The same amount of RNA was retro-transcribed and analyzed by means of qPCR using specific primer pairs for each NR (Table S2) In vivo fat body NR transcript expression levels were standardized by total RNA input, because the fat body is a dynamically developing tissue both before and following a blood meal, thus precluding the use of a FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al Nuclear receptors in Aedes aegypti PV 250 B PBM 20E (pg/female) 20E (pg/female) A 200 150 100 50 0d 120 4d0h 6h 12 h 24 h 36 h 48 h 100 50 0d a 1200 b b b b b b 2500 AaEcR-B 48 h 72 h b b b b b b b b AaHR3 2000 a a a a a a 1500 a a a 1000 a 500 0 AaUSP-A a 40 30 b 20 10 b b b b b b Relative mRNA (+SEM) Relative mRNA (+SEM) 36 h a 400 b 2500 ab b b 2000 1500 a 1000 300 b 200 100 300 AaUSP-B b AaHR4 b 0 25 24 h ab 800 50 12 h AaE75A 40 60 4d0h 6h 1600 60 150 AaEcR-A 80 PBM 200 72 h 100 20 PV 250 b b b b b AaE78 250 20 a 15 200 a 150 10 a a a a a a 100 a 50 a a a a 4d 6h 1d 4d PV 6h 12 h 24 h 36 h 48 h 72 h PBM 1d PV a a 12 h 24 h 36 h 48 h a 72 h PBM Fig Expression patterns of A aegypti nuclear receptors (NRs) in female fat bodies during vitellogenesis, and whole-body ecdysteroid titers are presented for comparison Data for the whole body ecdysteroid levels are from Hagedorn et al [111] and are expressed as pg per female For transcript analysis, equal amounts of total RNA from staged adult females were analyzed by RT-PCR The time points analyzed were 1–2 and 4–5 days pre-vitellogenic (PV), and 6, 12, 24, 36, 48 and 72 h after a blood meal (PBM) The profiles of the ecdysone receptor components AaEcRA, AaEcRB, AaUSP-A, AaUSP-B (A), ecdysone response genes AaE75A, AaHR3, AaHR4, AaE78 (B), AaHR39, AaHR78 (C), the competence factor AabFTZ-F1 isoforms A and B (C), the hepatocyte nuclear factor isoforms AaHNF-4A, AaHNF-4B, AaHNF-4C (D), the non-20E-responsive genes AaERR (D), AaTll, AaPNR-like (E), AaSvp, AaHR38 (F) and the housekeeping genes AaS7 (E) and AaActin (F) are expressed as relative mRNA and are the mean of three independent biological replicates The vertical bars indicate the SEM Means were separated using Tukey–Kramer HSD with time points sharing the same letter determined not to be significantly different (P £ 0.05) FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1239 Nuclear receptors in Aedes aegypti PV 250 D PBM 20E (pg/female) 20E (pg/female) C J Cruz et al 200 150 100 50 4d0h 6h 0d 50 12 h 24 h 36 h 48 h a a 24 h 36 h 48 h 72 h a 2000 a a a a 1000 b 10 000 AaHR78 b b b b b AaHNF4B 8000 a a a a 2000 AaβFTZ-F1A 100 a ab 60 ab 40 ab 20 b b ab 4000 a a ab Relative mRNA (+SEM) a a a 6000 a Relative mRNA (+SEM) 12 h 3000 a a 80 4d0h 6h a 120 50 4000 10 100 5000 AaHNF4A 30 10 150 0d AaHR39 PBM 200 72 h 40 20 PV 250 b b b 3500 AaHNF4C 3000 2500 a 2000 1000 25 AaβFTZ-F1B 50 20 15 b b b b ab b 40 30 a a a a a a a a 10 4d PV 6h 12 h 24 h 36 h 48 h 72 h PBM a a 1d a 20 a a AaERR a a 10 a 1500 500 b b b 1d 4d PV 6h 12 h a 24 h 36 h 48 h 72 h PBM Fig (Continued) ‘normalizing’ transcript The time points chosen for the current study address the complete vitellogenic cycle: pre-vitellogenesis (1–4 days pre-vitellogenesis), vitellogenesis (6–30 h PBM), early post vitellogenesis (36–48 h PBM) and late post vitellogenesis (72 h PBM), with these progressions including, respectively, active ribosomal biogenesis, massive protein synthesis, 1240 tissue autophagy and ribosomal biogenesis again [2,45,46] This developmental course can be observed through the dynamic expression profile of the commonly used ‘housekeeping’ transcript ribosomal protein S7 [47] (Fig 3E), as well as actin (Fig 3F) Such a condition is not applicable to the in vitro experiments, because all fat bodies used in these studies were at the FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al Nuclear receptors in Aedes aegypti F PV 250 PBM 20E (pg/female) 20E (pg/female) E 200 150 100 50 0d 400 300 4d0h 6h 12 h 24 h 36 h 48 h PV 250 PBM 200 150 100 50 72 h 0d 4d0h 6h AaTll 24 h 36 h 48 h 72 h 80 AaSvp a 12 h 60 a 200 40 20 b 10 b b b b 20 b b 140 AaPNR-like 120 100 80 60 a a a a a a a 40 20 a 16 14 12 10 a a a a a a a a AaS7 Relative mRNA (+SEM) Relative mRNA (+SEM) a 140 AaHR38 120 100 a 80 60 a 40 20 a a a 250 a AaActin 200 a 150 a ab ab 100 b b b b b b b 50 ab b b b 6h 12 h 24 h b 1d PV 4d 6h 12 h 24 h 36 h 48 h 72 h 1d PBM 4d PV 36 h 48 h 72 h PBM Fig (Continued) same developmental stage, hence the use of ribosomal protein S7 transcripts as the ‘housekeeping’ normalizer (no statistical change in expression levels for AaS7; J Cruz & A S Raikhel, unpublished observations) Following the in vivo time-course study, we wanted to determine whether the steroid hormone 20E might be responsible for the expression profile observed in vivo To this end, we carried out two different in vitro experiments First, our aim was to establish those NRs directly induced by 20E (primary-response genes) The fat bodies were incubated in the presence of 20E alone, cycloheximide (Chx) alone, 20E plus Chx or control media for h In the second experiment, our aim was to determine the NR transcripts that require an initial exposure to 20E followed by its withdrawal for induction (secondary-response genes) In this second experiment, the fat bodies were incubated in media supplemented with 20E for h, washed and then incubated for 12 more hours in a hormone-free medium As a control, fat bodies were incubated with or without 20E RNA extracted from these samples was analyzed by means of qPCR, and transcripts were normalized over AaS7 A summary of the 20E inducibility of the transcripts analyzed is presented in Table The A aegypti ecdysone receptor Two EcR isoforms (AaEcRA and AaEcRB) and two USP isoforms (AaUSP-A and AaUSP-B) have been characterized previously in A aegypti [48–50] The FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1241 Nuclear receptors in Aedes aegypti J Cruz et al expression pattern of AaEcRA followed the peak in 20E titers, as previously reported [48] The level of AaEcRA transcript increased slightly at 12 h PBM, reaching the maximum at 24 h (Fig 3A), an h delay if compared with the previously published pattern [48] By 36 h PBM, AaEcRA levels had declined significantly, reaching the pre-vitellogenic level (Fig 3A) There was no obvious peak in the expression profile of AaEcRB mRNA, but a slight increase shortly after the blood meal was observed, beginning to decline at the peak ecdysone titer, and once again to increase at the end of the vitellogenic period (48–72 h, Fig 3A) The patterns for AaEcRA and AaEcRB corroborate that previously reported [48]; however, using our qPCR approach, the fluctuations in AaEcRB mRNA abundance are not statistically significant Previous analysis of AaEcR transcript regulation by 20E, using an in vitro fat body culture system, suggested that the transcripts for both AaEcR isoforms are upregulated by 20E AaEcRA transcription required continuous presence of the hormone [48] (Fig 4A), whereas AaEcRB required the presence of 20E along with the translation inhibitor Chx [48] (Fig 4A) A short exposure of 20E has also been shown to induce AaEcRB transcription [48], an observation we could not repeat in our current study (Fig 4A) The two isoforms of AaUSP displayed distinct mRNA expression profiles during the vitellogenic period (Fig 3A) AaUSP-A mRNA abundance was at its maximum level the first day after the adult molt, as previously observed [49], and more descriptively, in agreement with the high abundance reported by Margam et al [47] at the end of the pupal stage Using our qPCR approach, however, the reported peak at 36 h PBM [49] was not observed The level of AaUSP-B transcript was relatively constant throughout the preand vitellogenic periods, with a slight increase beginning at the end of the vitellogenic period (48–72 h; Fig 3A) Previous 20E transcriptional regulation analysis in this laboratory, using semi-quantitative RTPCR, showed that AaUSP-A mRNA was upregulated after a short exposure to 20E and its withdrawal [49], a response that we could not corroborate using qPCR (Fig 4A) Furthermore, AaUSP-A mRNA levels only increased significantly when incubated with Chx in the absence of 20E (Fig 4A) By contrast, AaUSP-B transcripts were upregulated by 20E in combination with Chx, but also after a long exposure to 20E alone [49] Our current results, along with previous reports that have established a lack of fluctuation in the protein levels of AaEcR and AaUSP isoforms during vitellogenesis [13], suggest a lack of significant transcriptional and translational regulation of the two components 1242 that make up the ecdysone receptor Moreover, co-immunoprecipitation experiments using nuclear extracts of vitellogenic fat bodies of A aegypti demonstrated that the formation of the heterodimer AaEcR ⁄ AaUSP can be regulated by other NRs through protein–protein interactions AaHR38 and AaSvp, during the arrest and termination of vitellogenesis, respectively, bind to AaUSP preventing its heterodimerization with AaEcR, and, consequently, the 20Edependent activation is blocked The presence of 20E, however, favors the formation of the AaEcR ⁄ AaUSP heterodimer [13], indicating that the regulation of the activity of these proteins occurs through protein–protein interactions, as well as ligand-mediated switch NR transcripts regulated by 20E The Aedes E75 NR family has three isoforms, each with a distinctive N-terminal A ⁄ B domain [36] AaE75B cannot bind DNA due to the lack of one of its zinc fingers All three isoforms present a similar pattern of expression and regulation by 20E [36]; thus, we only present the data corresponding to the AaE75A isoform As in D melanogaster [51] and Manduca sexta [52], AaE75A is expressed transiently very early in the ecdysone-induced regulatory cascade and is directly regulated by 20E [36], response characteristics that define it as an early-gene Expression of AaHR3 and AaHR4 occurred only after that of AaE75A reached its peak (Fig 3B) AaHR3 exhibited a sharp increase in transcript abundance peaking at 24 h PBM [37], whereas AaHR4 mRNA levels remained much more flat (Fig 3B) In in vitro fat body culture, AaHR3 transcription was activated by 20E, but this upregulation only became significant if the tissue was incubated simultaneously with 20E and Chx, or was continuously exposed to the hormone for 16 h [37] AaHR4 transcription was significantly upregulated by combining 20E and Chx in the culture medium (Fig 4B), but the presence of 20E alone also increased the transcript abundance, although not significantly (Fig 4B) The primary difference between the observed transcript profiles of AaHR3 and AaHR4 is that AaHR4 is significantly abundant in newly eclosed female fat bodies (pre-vitellogenic day, Fig 3B), suggesting a possible role in the transition between pupae and adult It is generally believed that gene transcripts induced in vitro only in the presence of 20E and the protein synthesis inhibitor Chx, as observed for AaHR4 and AaHR3, are negatively repressed by 20-induced genes (e.g the early response genes) [53,54] M sexta GV1 cell transfection assays demonstrated FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al A 16 Nuclear receptors in Aedes aegypti AaEcRA 16 ** 14 12 10 8 6 4 2 AaEcRB 18 16 14 12 10 AaUSPA 25 * AaUSPB 1800 1600 1400 1200 1000 800 600 400 200 AaEcRB 18 16 14 12 10 AaUSPA 25 AaUSPB Relative mRNA (SEM) 1800 1600 1400 1200 1000 800 600 400 200 Relative mRNA (SEM) ** 12 * 10 AaEcRA 14 ** ** 20 15 15 10 10 5 *** 20 6 AaE75A **** AaE75A 4 3 ** ** ** 1 0 CM 0h CM 20E Chx 20E + Chx 6h CM 0h CM 20E 4h CM 20E h20E + CM 16 h Fig Effect of 20E on the transcription of A aegypti nuclear receptors (NRs) in isolated pre-vitellogenic (PV) fat bodies In the first experiment (left panel), PV fat bodies dissected from female mosquitoes (5 days) were incubated in culture media (CM) for 30 and established as initial time point (0 h); then were incubated for h in CM, with 10)6 M 20E (20E), 10)5 M Chx (Chx) or 20E and Chx together (20E + Chx) In the second experiment (right), day PV fat bodies were incubated in CM for 30 and established as initial time point (0 h); then were incubated in CM or with 20E for and 16 h, or with a pulse treatment of 20E for h then removal of 20E, followed by incubation within CM for an additional 12 h (4 h 20E + CM) At the indicated time points, a group of nine fat bodies were collected and RNA levels were analyzed using qPCR Transcript abundance values for AaEcRA, AaEcRB, AaUSP-A, AaUSP-B, AaE75A (A), AaHR3, AaHR4, AaE78, AaHR39, AaHR78 (B) and AabFTZ-F1A, AabFTZ-F1B, AaHNF-4A, AaHNF-4B, AaHNF-4C (C) are presented as mRNA quantity normalized against ribosomal protein S7 transcripts and represent the mean of three independent biological replicates The vertical bars indicate the SEM Each normalized transcript was compared against the h CM using Dunnett’s method Asterisks indicate statistically significant differences at ****P £ 0.0005; ***P £ 0.005; **P £ 0.05; *P £ 0.1 levels FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1243 Nuclear receptors in Aedes aegypti B J Cruz et al **** AaHR3 AaHR3 ** ** 2 0 14 14 *** AaHR4 12 AaHR4 13 10 1400 AaE78 Relative mRNA (SEM) Relative mRNA (SEM) ** 1200 1000 800 600 * 400 200 10 AaE78 1200 1000 800 600 ** 400 200 10 **** AaHR39 1400 AaHR39 6 * 4 2 0 6 ** AaHR78 4 3 2 AaHR78 0 CM 0h CM 20E Chx 20E + Chx CM 6h 0h CM 20E 4h CM 20E h 20E + CM 16 h Fig (Continued) that MsE75A represses the induction of MsHR3 by binding to the consensus monomeric response element (A ⁄ T-AGGTCA) present in its promoter [55] Furthermore, MsHR4 transcripts have been shown to appear only after MsHR3 mRNA and MsHR3 protein decline to low levels both in vivo and in vitro [56] Finally, the transcription of MsHR4 is induced by 20E only in GV-1 cells that have been transfected with MsHR3 1244 dsRNA [57]; these authors suggested a cascade activation model in which MsHR3 is one of the 20E-induced factors inhibiting MsHR4 expression and, in turn, MsHR4 would act as a transcriptional repressor of MsbFTZ-F1, a NR that appears sequentially after MsHR4 [58] In D melanogaster, the transcriptional cascade described for these NRs displays some differences from that observed for M sexta but, FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al C 140 Nuclear receptors in Aedes aegypti 140 AaβFTZ-F1A **** 120 100 AaβFTZ-F1A 130 *** 40 80 60 20 40 20 0 400 400 AaβFTZ-F1B ** 300 300 ** 200 200 * 100 0 50 Relative mRNA (SEM) 100 Relative mRNA (SEM) AaβFTZ-F1B AaHNF4A ** 40 30 20 10 12 50 * 10 *** 30 *** 20 10 12 AaHNF4B AaHNF4A 40 AaHNF4B 10 8 6 4 2 0 200 200 AaHNF4C 160 120 80 80 40 * 160 120 AaHNF4C 40 0 CM 0h CM 20E Chx 20E + Chx CM 6h 0h CM 20E 4h CM 20E h 20E + CM 16 h Fig (Continued) importantly, are involved in coordinating the cascade of expression of various NRs In D melanogaster, DmE75B represses DmHR3-dependent transactivation through protein–protein interaction [59] DmHR3 and DmHR4 act together to induce DmbFTZ-F1 expression and, as the ecdysteroid titer declines, repress the transcription of the early 20E-induced genes such as DmEcR, DmE74A and DmE75A [60,61] Hence, DmHR3 and DmHR4 function as a switch that defines the larval–prepupal transition by arresting the early regulatory response to ecdysone at puparium formation, thus facilitating the induction of the DmbFTZF1 competence factor in mid-prepupae As should be evident, the cascade of transcription factor activation during the decline of the ecdysteroid titer is complex, involving not only 20E, but also the interplay among FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1245 Nuclear receptors in Aedes aegypti J Cruz et al the ecdysteroid-induced transcription factors Such cascades will likely differ among species, or even physiological processes In A aegypti, the discrete expression of both AaHR3 and AaHR4 suggests their function in the regulatory hierarchy in the mosquito fat body during vitellogenesis Indeed, the analysis of the upstream region of the gene encoding AaHR3 has revealed the presence of two regions rich in putative binding sites for both EcR-USP and E75 (nubiscan v 2.0, http:// www.nubiscan.unibas.ch/; Cruz & Raikhel, unpublished observation), suggesting a similar regulation of this NR as reported for other insect species Furthermore, analysis of the AaVg gene regulatory regions has identified an E75 binding site within a region required for high levels of AaVg expression [62] Because this response element is shared by E75 and HR3 [63,64], as well as considering the relative timing of AaE75 and AaHR3 expression, we propose that these NRs could compete for their common response element on the AaVg promoter acting in the regulation of AaVg synthesis and termination The NR AaE78 mRNA presented a limited peak at 36 h PBM, when the ecdysone levels were low (Fig 3B), as opposed to the apparent induction by 20E in vitro (Fig 4B) In D melanogaster, DmE78 encodes at least two protein isoforms from distinct promoters [65] DmE78 mutations have no effect on viability or fertility, although this lesion does have an apparent effect on chromosomal puffing [66] Based on the first assembly of the A aegypti genome, only one AaE78 protein isoform has been predicted (Table 1), but further studies are required to determine whether there may be different isoforms in A aegypti, along with their possible roles in A aegypti reproduction Transcript levels of AaHR39 were highest after eclosion and in post-vitellogenic fat bodies, and lowest during vitellogenesis (Fig 3C) 20E was capable of inducing the transcription of AaHR39, with a much more significant increase in the presence of Chx (Fig 4B) This finding agrees with that observed in vivo, in which transcript abundance increased later in the vitellogenic cycle HR39, a NR with high sequence similarity to FTZ-F1, has been identified in other insect species, D melanogaster, B mori and T castaneum [21,67–69] In D melanogaster, both DmbFTZ-F1 and DmHR39 mRNAs are expressed during the same developmental stages; however, the expression of DmHR39 typically precedes DmbFTZ-F1 and seems to be downregulated when DmbFTZ-F1 reaches its maximum levels [7,70] Both have similar DNA-binding domains, and DmHR39 represses the transcription activated by DmbFTZ-F1 through bind1246 ing to the same response element [67,71] Moreover, a GAL4-LBD ‘ligand sensor’ system showed that DmHR39 does not display detectable activation at any stage of development, suggesting that this NR acts as a repressor [72] It would be interesting to determine whether the reciprocal patterns of expression between AaHR39 and both AabFTZ-F1 isoforms during the vitellogenic period are of functional significance The period during which AaHR78 displayed its maximum levels correlates with the pre-vitellogenic preparatory period in the fat body, being low with higher titers of 20E and gradually rising again after 36 h PBM when 20E levels began to decline (Fig 3C) The acquisition of competence in the fat body is manifested by several cellular events, such as development of the endoplasmic reticulum and Golgi complexes and ribosome proliferation [2,45] In D melanogaster, DmHR78 is required for growth and viability during larval stages, as demonstrated by DmHR78-null mutants displaying growth defects, and dying as small L3 [73] Although the mechanisms that underlie the biological function remain unknown, it has been shown that DmHR78 can inhibit the ecdysone-dependent induction of a reporter gene through binding to a subset of EcR ⁄ USP-binding sites in vitro [74,75] A recent study demonstrated that DmHR78 activity is controlled by a co-repressor, Moses, and the balance between these two proteins determines Drosophila growth rate [76] In A aegypti, the maximum AaHR78 transcript abundance in the fat body coincides with the growth and remodeling period in this tissue, suggesting that AaHR78 may be involved in this process Expression and regulation of the A aegypti competence factor, bFTZ-F1 The A aegypti competence factor, bFTZ-F1, was first reported as a unique transcript [23], and the in vivo expression of its transcripts and 20E regulation was conducted with a primer pair located in the LBD, a region common to both isoforms identified during the current study [15,23] However, the sequence chosen to generate an antibody against AabFTZ-F1 was the A ⁄ B-specific region of AabFTZ-F1A [15] As a result, the experiments conducted using this antibody are specific for AabFTZ-F1A isoform For the current study, we determined whether the two isoforms displayed different expression profiles and are both regulated by 20E in a similar manner As shown in Fig 3C, the level of AabFTZ-F1A transcript abundance was significantly higher than that of AabFTZF1B in the fat bodies of newly eclosed females After FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al the onset of vitellogenesis, AabFTZ-F1A dropped dramatically, remaining at low levels until vitellogenesis was complete (36–72 h PBM) AabFTZ-F1B mRNA displayed near background levels of transcript abundance during pre-vitellogenic development and throughout vitellogenesis, with expression levels only beginning to rise following vitellogenesis (36 h PBM; Fig 3C) Experiments with in vitro fat body cultures revealed that AabFTZ-F1A expression is not directly under 20E regulation (Fig 4C), and its transcript levels only reached significant quantities when exposed to the protein synthesis inhibitor Chx This observation could be explained by a stabilization of pre-existing mRNAs [53,54,77] or due to Chx inhibiting the expression of repressor factors In D melanogaster, the NR DmbFTZ-F1 has been defined as a ‘competence’ factor due to the requirement for its expression during mid-prepupal development, allowing for the correct response to ecdysone at the end of the pupal stage [78–80] Broadus et al [79] hypothesized that DmbFTZ-F1 may have direct interaction with target promoters, but the molecular mechanism remains unclear In A aegypti, however, studies within our laboratory have been carried out to determine the role of AabFTZ-F1A in the acquisition of competence in the pre-vitellogenic fat body (i.e the ability of the fat bodies to respond to 20E) By contrast to D melanogaster, where the acquisition of competence is regulated by 20E, in A aegypti this preparatory period is regulated by JH III [2,81] It was determined that JH III coordinates the development of competence through its ability to promote the translation of AabFTZ-F1A gene [15] With the gained competence in the fat body, a blood meal initiates a cascade in which AabFTZ-F1A promotes ecdysone receptor activity through binding the Vg promoter and recruiting the coactivator p160 ⁄ SRC (AaFISC), which acts as a bridge between AabFTZ-F1A and AaEcR ⁄ AaUSP heterodimers, establishing a functional multiple protein complex on the Vg promoter [16] Thus, the DNA binding and protein interaction provide a combinatorial code required for specific gene activation by 20E By contrast to AabFTZ-F1A, AabFTZ-F1B displayed a different response to 20E Withdrawal of 20E from the fat body culture after an initial h incubation in the presence of 20E resulted in a considerable elevation of AabFTZ-F1B mRNA (Fig 4C), as previously found analyzing the common region [23] and in agreement with studies of D melanogaster [78], B mori [82] and M sexta [56] In D melanogaster, DmHR3 activates DmbFTZ-F1 mRNA expression through a response element in the promoter region of the Nuclear receptors in Aedes aegypti DmbFTZ-F1 gene [59,60,83] But DmE75B, which lacks a complete DNA-binding domain, inhibits this inductive function by forming a complex with DmHR3 on the DmbFTZ-F1 promoter This mechanism provides specific timing for DmbFTZ-F1 transcription that requires the presence of 20E for DmHR3 induction, but its withdrawal for the disappearance of DmE75B [59,60] In vivo, maximum expression of AabFTZ-F1B occurred after peak AaE75 and AaHR3 expression, suggesting a similar regulatory activation cascade in the vitellogenic mosquito fat body (Fig 3B,C) Further studies are necessary to clarify the regulation of AabFTZ-F1B and its possible involvement in the cascade as ecdysteroid titers decline later in the vitellogenic cycle Expression and 20E regulation of A aegypti HNF-4 isoforms There are three isoforms of the HNF-4 in A aegypti, which have been previously designated AaHNF-4A, AaHNF-4B and AaHNF-4C [17] The A and B isoforms are typical members of the NR family, differing only in the N-terminal end of the variable A ⁄ B domain The third mosquito isoform, AaHNF-4C, lacks the greater part of this A ⁄ B domain and the complete DBD; consequently, it cannot bind DNA [17] The expression profiles of these three AaHNF-4 isoforms differ over the course of the vitellogenic cycle in the female fat body AaHNF-4A and AaHNF-4C were barely detectable during the pre-vitellogenic period, only beginning to increase after vitellogenesis (36 h PBM) and reaching a significant level by 48–72 h PBM (Fig 3D) By contrast, AaHNF-4B mRNA was highly upregulated days after eclosion, and quickly dropped after a blood meal, beginning to rise again after vitellogenesis (36–72 h PBM; Fig 3D) Not surprisingly, the three isoforms also displayed distinct responses to 20E exposure Of unique interest, AaHNF-4A mRNA was upregulated in hormone- and Chx-free medium after 4, or 16 h incubation periods (Fig 4C) AaHNF-4B transcript showed no response to 20E, and AaHNF-4C mRNA a secondary response, as demonstrated by its significant upregulation after a short exposure to the hormone followed by its withdrawal (Fig 4C) In vertebrates, HNF-4 has an essential role in hepatocyte differentiation and lipid homeostasis [84,85] Mutations in the human HNF-4a cause a type II diabetes called maturity-onset diabetes of the young, subtype (MODY1), which is associated with defective glucose-dependent insulin secretion from pancreatic beta cells [86] HNF-4a activates the insulin gene FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1247 Nuclear receptors in Aedes aegypti J Cruz et al directly [87], but also plays a crucial role in the transcriptional regulation of hepatic gluconeogenic enzymes that are activated at fasting and suppressed in a fed state [88] In D melanogaster, a recent study using an in vivo ligand-detection system that follows NR LBD activation patterns in vivo, by way of a GAL4-LBD system [89], showed that the activity of GAL4-DmHNF-4, along with GAL4-DmHR3 and GAL4-DmHR38, in fat body is dramatically downregulated at puparium formation This coincides with the cessation of feeding that occurs at the end of the larval development [72] Thus, it was concluded that these NRs might respond to nutrients or metabolites In A aegypti, all three AaHNF-4 isoforms are upregulated at the end of the vitellogenic period, a time when the female has fully digested and processed the blood meal, and once again begins a fasting period until the next blood feed By contrast to what is observed in vivo, the in vitro experiments describe a completely different effect, where AaHNF-4C is slightly upregulated with a h ecdysone pulse, placing it as a secondary 20E response gene; AaHNF-4B is only upregulated in the presence of Chx, suggesting a transcript stabilization effect of this protein inhibitor; and, finally, the very surprising result for AaHNF-4A We observed a strong upregulation after incubation in culture medium for 4–16 h (Fig 4C) In our laboratory, it has been demonstrated that the blood-meal-dependent signal that triggers the transcriptional activation of AaVg is regulated by both the 20E regulatory pathway and an amino acid dependent pathway [90,91] Hansen et al [90] demonstrated that the TOR pathway is indeed transmitting the amino acid signal to the Vg gene in the mosquito fat body The in vitro activation displayed in AaHNF-4A in hormone-free media that harbors a complete balance of amino acids suggests the involvement of amino acids in its regulation This amino acid induced signal requires protein translation, as demonstrated by the lack of upregulation of AaHNF-4A in the presence of Chx (Fig 4B) Addressing the question of the nature of this signal and its importance in the fat body metabolic status will reveal interesting information regarding the dynamics of the fat body and provide the opportunity to use the mosquito fat body as a model system for deciphering the function of NRs in different metabolic pathways NR transcripts not affected by 20E In the female fat body, there were two periods during which expression of AaERR mRNA levels was higher: in newly eclosed and at 36–72 h PBM (Fig 3D), a time when the blood digestion is complete and mobili1248 zation of nutrient reserves from the fat body begins Transcript levels of AaERR were not affected by any treatment in vitro (data not shown) Vertebrates encoded three ERR isoforms: a, b and c [92–94], all of which share homology with estrogen receptors, but not bind to estrogen or other natural ligands [95] The GAL4-LBD system showed that the LBD of Drosophila ERR displayed a remarkable switch in activity during mid-embryogenesis and in the mid-third instar, suggesting that its activity is modulated by one or more ligands, although the nature of that remains undetermined [72] Several lines of evidence have suggested a role for vertebrate ERRa and ERRc in the control of metabolic genes Both isoforms are involved in the regulation of hepatic pyruvate metabolism, specifically inhibiting glycolytic flux through regulation of key enzymes in the oxidation of glucose to acetylCoA This family of NRs act synergistically with the peroxisome proliferator-activated receptor c coactivator (PGC1-a) and forkhead transcription factor (FoxO1), blocking the conversion of pyruvate to acetyl-CoA in the mitochondria, while insulin suppresses its effect [96] AaTll mRNA was highly expressed in newly eclosed female fat bodies, sharply decreased to minimum levels by day pre-vitellogenesis, and remained low during the whole vitellogenic period, with only small non-significant fluctuations observed (Fig 3E) Such a lack of fluctuation with known ecdysone titers in vivo is in agreement with the lack of effect of any in vitro treatment on its expression levels (data not shown) In D melanogaster, Tll NR acts as a gap gene during the early steps of embryogenesis and is involved in controlling terminal genes that result in normal development of head and posterior structures [97,98] Later in embryonic development, Tll is also necessary for the establishment of the D melanogaster embryonic visual system as well as for the development of the most anterior region of the brain [99,100] Tlx, the vertebrate ortholog of Tll, is required for the correct development of the visual system and neurogenesis [101,102] The expression of the NR AaPNR-like was constant within the adult female fat body (Fig 3E) and no regulation by 20E was demonstrated in the cultured fat body in vitro (data not shown) This NR has been recently identified in the Ap mellifera genome, and in situ hybridization studies revealed the presence of this transcript in a small number of cells in the developing eye [20] This NR and other members of the subfamily NR2E (Tll, HR51, Dsf and HR83) are expressed strictly within specific regions of the CNS or the visual system, which is in complete agreement with FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS J Cruz et al their putative functions Taken together, these data support our hypothesis that our observed amplification of AaTll and AaPNR-like from the mosquito fat body corresponds with a basal level of transcription, as previously discussed for AaHR51 and AaDsf AaSvp transcript abundance was high in the fat body of newly eclosed females, although not significantly different from that at other time points examined (Fig 3F), in agreement with previous reports for AaSvp [103] AaHR38 mRNA also displayed its maximum level in the fat body of newly eclosed females, remaining low thereafter, with the exception of a small peak at 36 h PBM (Fig 3F) Previous analysis of AaHR38 displayed a similar pattern, but with a small peak observed 24 h PBM [104] Not surprisingly, neither NR was affected by any in vitro treatment (data not shown) Furthermore, it has been shown that both proteins were reported to act as repressors of the AaEcR ⁄ AaUSP heterodimer through protein–protein binding with AaUSP [13], thus preventing formation of the functional ecdysone receptor and consequently inhibiting AaVg expression AaHR38 sequesters AaUSP during the pre-vitellogenic period while AaSvp operates during the termination period [13] In summary, this study provides a general overview of the complete family of A aegypti NR genes expressed during the vitellogenic period, a period important not only for the events that will provide nourishment for the developing oocyte, but also for a wide variety of metabolic processes A aegypti is an anautogenous mosquito and an extremely efficient disease vector because it requires host contact This understanding of the molecular regulation of vitellogenesis is important to achieve significant advances in the development of future vector- and vector-borne, disease-control strategies Experimental procedures Annotation of A aegypti NRs A set of amino acid sequences corresponding to the 21 NRs identified in D melanogaster (FlyBase Source; http:// flybase.bio.indiana.edu) were used to search for orthologs in A aegypti (http://aaegypti.vectorbase.org/index.php) using the BLASTP tool against the predicted protein dataset of A aegypti The sequences gleaned from the A aegypti database were examined manually through multiple sequence alignment using clustalw against previously identified orthologs in other insect species (sequences obtained from NCBI) Our in silico analysis predicted that some of the putative A aegypti NR sequences were present in unassembled predicted transcripts, while others contained Nuclear receptors in Aedes aegypti erroneous exon predictions relative to their orthologs in other species To address this issue, we validated our predictions using 5¢-RACE and RT-PCR analysis Phylogenetic analysis In order to classify and analyze phylogenetic consistency, a tree of all identified A aegypti NRs was created NR sequences used in the analyses were obtained from different sources: GenBank (http://www.ncbi.nlm.nih.gov/) for D melanogaster, Ap mellifera and Homo sapiens, VectorBase (http://www.vectorbase.org/) for Anopheles gambiae, and Beetlebase (http://www.bioinformatics.ksu.edu/Beetle Base/) for T castaneum The accession numbers for all NRs are available in Table S1 Phylogenetic analysis was performed on protein sequences aligned using t-coffee [105] with default parameters The phylip suite of programs [106] was used to create a Neighbor-joining tree and to estimate nodal support using 100 bootstrap replicates Branch lengths on the majority-rule consensus tree were estimated using tree-puzzle [107] Animals Mosquitoes were raised as described previously [108] Larvae were fed a standard diet [109], and adults were fed on 10% sucrose continuously by wick Adult females 3–5 days after eclosion were allowed to feed on anesthetized white rats to initiate vitellogenesis All dissections were performed in A aegypti physiological saline at room temperature [1] Expression of identified NRs in the fat body and ovaries of adult females following a blood meal To determine whether the identified and annotated NRs are expressed in a temporal manner in two reproductive tissues of adult female A aegypti, RT-PCR analysis was conducted against abdominal walls with adhering fat bodies (hereafter referred to as the fat body) and ovaries before vitellogenesis (pre-vitellogenic), at its onset (6 + 12 h PBM) and at its peak (18 + 24 h PBM) Total RNA was extracted from fat bodies and ovaries of 10 females using the TRIzol method (Invitrogen, Carlsbad, CA, USA) The isolated total RNA was subsequently cleaned using RNeasyÒ mini kit columns (Qiagen, Valencia, CA, USA), which included an on-column DNase I digestion (Qiagen) cDNA was synthesized from 2.5 lg of the DNase I-treated RNA using Superscript II (Invitrogen) To standardize RT-PCR inputs, a master mix containing HotStarTaq PCR Master Mix (Qiagen) and forward and reverse primers (final concentration ⁄ PCR = 100 nm each; see Table S2 for primer sequences) was prepared and aliquoted; to this, cDNA of the different tissues and time points was added The samples were subjected to PCR FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1249 Nuclear receptors in Aedes aegypti J Cruz et al amplification with a number of cycles within the linear range of amplification, preincubation at 95 °C for 15 followed by 30–40 cycles, depending on the NR (95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s) and a final elongation at 72 °C for Expression of NRs in adult female fat body following a blood meal Total RNA was extracted from fat bodies, column purified, which included a DNase I treatment, and cDNA synthesized as described above The time points analyzed were 1–2 and 4–5 days pre-vitellogenesis, and 6, 12, 24, 36, 48 and 72 h PBM To standardize qPCR inputs, a master mix that contained iQ SYBR Green Supermix and forward and reverse primers was prepared (final concentration = 100 nm per qPCR; see Table S2 for primer sequences) The master mix was then aliquoted into iCycler iQÔ PCR plates (Bio-Rad, Hercules, CA, USA), and cDNA was subsequently added at 400 ng total RNA input per qPCR All samples were analyzed on the iCycler iQ Real Time PCR Detection System (Bio-Rad) Standards were generated using a serial dilution of cDNA preparations known to contain a high concentration of the transcripts analyzed Samples from three biological replicates were analyzed, and their means separated by means of TukeyKramer HSD (P £ 0.05) (jmp statistical discovery software from SAS Institute Inc., Cary, NC, USA) In vitro fat body culture Fat bodies were dissected from 4- to 5-day-old pre-vitellogenic females and incubated in an organ culture system, as previously described [110] Three sets of three fat bodies were cultured as described, pooled following the incubation, and subsequently processed for total RNA and cDNA synthesis, as described above To determine whether 20E promotes NR transcription within the fat body, the dissected fat bodies were incubated for 30 in culture medium without hormone, followed by a h incubation in the presence or absence of the hormone (10)6 m 20E) To test the effect of the protein synthesis inhibitor Chx on the expression of previously uncharacterized A aegypti NRs and 20E primary response genes, the fat bodies were pretreated with culture medium containing 10)5 m Chx for 30 min, then incubated with Chx either with or without 20E for an additional h A second experiment addressed the effect of an initial induction by 10)6 m 20E followed by removal of the said hormone; this was accomplished by first providing 10)6 m 20E for h, followed by washing the fat bodies three times with hormone-free medium and maintaining the fat bodies in hormone-free media for an additional 12 h The comparable control was the maintenance of fat bodies for 16 h in media with or without 10)6 m 20E As a control for all 1250 experiments, the fat bodies were incubated in hormone-free medium supplemented with 10% ethanol (the 20E carrier) Samples from three biological replicates were analyzed using qPCR, relative transcripts 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