Tài liệu Báo cáo khoa học: Nuclear receptors in the mosquito Aedes aegypti Annotation, hormonal regulation and expression profiling ppt

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
Mosquitoes are vectors of some of the world’s most
devastating diseases. Malaria causes approximately
1 million deaths annually () and
dengue, a rapidly expanding disease in most tropical
and subtropical areas of the world, has become the
most significant arboviral disease of humans. Anophe-
line 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 under-
standing 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
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:
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 dis-
eases of humans and domestic animals. Understanding the molecular
mechanisms regulating mosquito egg development may contribute signi-
ficantly 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.
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
hormones governing vitellogenesis are the sesquiterpe-
noid juvenile hormone III (JH III) and the steroid 20-
hydroxyecdysone (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 vitello-
genesis [1,2]; 72 h is typically required after eclosion to
achieve this state. This development toward ‘compe-
tence’ 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 dis-
sected in detail in studies of Drosophila melanogaster
development [6–9]. The functional 20E receptor is com-
posed 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 expres-
sion of a set of genes, including hormonal receptor 3
(Hr3 or Hr46), HR4, HR39, E75, E78 and fushi tarazu
transcription factor 1 (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 aforemen-
tioned factors, together with EcR and USP, are mem-
bers 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 associ-
ating 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 acquisi-
tion of competence in the fat body during the first
3 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, AabFTZ-
F1 promotes EcR activity by recruiting the coactivator

p160 ⁄ SRC (AaFISC) which, in turn, binds the
AaEcR ⁄ AaUSP heterodimers, establishing a functional
multiple protein complex on the Vg promoter [16]. By
24 h PBM, AaVg transcript levels reach their maxi-
mum, after which they sharply decline, concluding
with the termination of vitellogenesis. In this termina-
tion process, mosquito Seven-up (AaSvp), a NR mem-
ber, plays a central role replacing AaUSP in the
AaEcR ⁄ AaUSP heterodimer complex, thereby block-
ing 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 Broad-
complex, 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 expres-
sion 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 mosqui-
toes 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. mela-
nogaster compared against the A. aegypti database
( We identi-
fied 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 mel-
lifera and Tribolium castaneum PNR-like NR [20–22].
Following our manual initial annotation, some of our
in silico predicted sequences were split into unassem-
bled automatic predicted sequences in the A. aegypti
genomic database; with some given splice sites and
exons not predicted in our original manual annotation.
Nuclear receptors in Aedes aegypti J. Cruz et al.
1234 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 1.

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. melano-
gaster, 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 fam-
ily members that lack the LBD. In D. melanogaster,
Table 1. 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 Isoform
Vector base
accession no.
NCBI
accession no.
20E
response
NR0A2 Knirps-like AaKnrl AAEL011231 BN001172 NT
NR1D3 Ecdysone-induced AaEip75B AaE75A AAEL007397 AM397060 P
protein 75B AaE75B AM397061 P
AaE75C AM397062 P
NR1E1 Ecdysone-induced
protein 78C
AaEip78C 24839.m08763,
AAEL000327
AM773442 P
NR1F4 Hormone receptor-like in 46 AaHr46 AAEL009588 AF230281 P
NR1H1 Ecdysone receptor AaEcR AaEcRA AAEL010719, AAEL009600 AY345989 P
AaEcRB AAEL010715, AAEL009600 AF305214 NR
NR1J1 Hormone receptor-like in 96 AaHr96 AAEL007202, AAEL007214 AM773443 NT
NR2A4 Hepatocyte nuclear factor 4 AaHnf4 AaHnf4-A AAEL011323 AF059026 NR
AaHnf4-B AF059027 NR
AaHnf4-C AF059028 S
NR2B4 Ultraspiracle AaUSP AaUSP-A AAEL000395 AF305213 NR
AaUSP-B VBTranscript 011769,
AAEL000395

AF305214 S
NR2D1 Hormone receptor-like in 78 AaHr78 AAEL001796 BN001173 P
NR2E2 Tailless AaTll AAEL003020 BN001174 NR
NR2E3 Hormone receptor-like in 51 AaHr51 AAEL007190 BN001175 NT
NR2E4 Dissatisfaction AaDsf AAEL005381 BN001178 NT
NR2E5 Hormone receptor-like in 83 AaHr83 AAEL007350 AM773444 NT
NR2E6 PNR-like AaPNR-like AAEL008043, AAEL008047 AM773445 NR
NR2F3 Seven up AaSvp AAEL006916, AAEL002765,
AAEL002768
AF303224 NR
NR3B4 Estrogen-related receptor AaERR AAEL013546 BN001176 NR
NR4A4 Hormone receptor-like in 38 AaHr38 AAEL013270 AF165528 NR
NR5A3 Ftz transcription factor AaFTZ-F1 AabFTZ-F1A AAEL002053, AAEL002062 AF274870 NR
AabFTZ-F1B AAEL002062 AM773446 S
NR5B1 Hormone receptor-like in 39 AaHr39 AAEL001304 BN001177 P
NR6A1 Hormone receptor-like in 4 AaHr4 AAEL005850, AAEL005864 AM773447 P
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1235
this group is composed of three members (knirps,
knirps-like and eagle). In A. aegypti, we only charac-
terized 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 canoni-
cal 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 Velav-
erde 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. 1 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. 1 and Table S1), indicating that the
classification proposed for the A. aegypti NRs is sup-
ported by phylogenetic consistency. The only discrep-
ancy 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
RT-PCR with a specific primer pair for each NR (see
Table S2 for primer sequences). Two biological repli-
cates were analyzed, and Fig. 2 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 sug-
gests 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 fluctu-

ation 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 tran-
scripts 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 stud-
ies 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 dis-
cernable using our RT-PCR analysis. By contrast to
this restricted expression observed within nervous
systems, HR96 has been implicated in regulating xeno-
biotic responses in D. melanogaster and is highly
expressed in tissues that monitor and metabolize xeno-
biotics, 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,
Nuclear receptors in Aedes aegypti J. Cruz et al.
1236 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 1. Phylogenetic tree of insect NRs. The different NR families are organized into groupings, NR1–NR6. The tree was constructed follow-
ing 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.
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1237
AaE75C and AaHR3 mRNAs increased with the
in vivo ecdysteroid peak, corroborating results from
previous studies [36,37]. Such an increase in expres-
sion levels within the ovary along with known ecdys-
teroid titers in vivo was also observed for AaEcRA,
AaEcRB, AaHR4, AaE78, AaHNF-4A and AaHNF-
4C 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
has been extensively studied in development and
metamorphosis in insects, its potential role in promot-
ing oogenesis has received significantly less attention.
EcR mutant females of D. melanogaster display
abnormal egg chamber development and loss of vitell-
ogenic egg stages [38] as well as chorion malforma-
tions [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
8 and 9 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 reiter-
ative 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 methodol-
ogy, 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 contri-
bution for later embryonic development. Indeed, nine
NR transcripts are maternally loaded in D. melanog-
aster ovaries [7].
Expression and 20E regulation of A. aegypti NRs
in the fat body
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 expres-
sion within the fat body in our earlier RT-PCR experi-
ment. 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
Fig. 2. 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 repre-
sentative of two biological replicates.
Nuclear receptors in Aedes aegypti J. Cruz et al.
1238 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
AaHR3
0
500
1000
1500
2000
2500
b
ab
a
a

b
b
b
b
AaHR4
0
300
1000
1500
2000
2500
a
b
b
b
b
b
b
b
AaE78
50
100
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300
a
a
a
a

a
a
a
a
0
100
200
AaEcR-A
0
20
40
60
80
100
120
b
a
b
b
b
b
b
b
0 h 6 h 12 h 24 h 36 h 48 h 72 h
20E (pg/female)
50
100
150
200
250

0 d
4 d
Relative mRNA (+SEM)
Relative mRNA (+SEM)
AaUSP-A
0
10
20
30
40
50
60
a
b
b
b
b
b
b
b
AaUSP-B
0
5
10
15
20
25
a
a
a

a
a
a
a
a
AaE75A
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
0
400
800
1200
1600
b
a
b
b
b
b
ab
b
PV PBM
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
PV PBM
PV
AB
PBM
AaEcR-B
0
1
2

3
4
5
6
7
a
a
a
a
a
a
a
a
0 h 6 h 12 h 24 h 36 h 48 h 72 h
20E (pg/female)
50
100
150
200
250
0 d
4 d
PV PBM
Fig. 3. 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).
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1239
‘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,
tissue autophagy and ribosomal biogenesis again
[2,45,46]. This developmental course can be observed
through the dynamic expression profile of the com-
monly used ‘housekeeping’ transcript ribosomal pro-
tein 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
AaERR
10
20
30
40
50
a
a
a
a
a
a

a
a
0
Relative mRNA (+SEM)
Rel
ative mRNA (+SEM)
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
PV PBM
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
PV PBM
AaHR39
0
20
30
40
50
a
a
a
a
a
a
a
a
AaHR78
0
2
4
6
8

10
a
a
a
a
a
a
a
a
10
AaHNF4B
0
2000
4000
6000
8000
10 000
b
a
b
b
b
b
ab
a
AaHNF4C
0
500
1000
1500

2000
2500
3000
3500
b
b
b
b
b
ab
a
a
AaHNF4A
0
1000
2000
3000
4000
5000
b
b
b
b
b
b
a
a
0 h 6 h 12 h 24 h 36 h 48 h 72 h
20E (pg/female)
50

100
150
200
250
50
100
150
200
250
0 d 4 d
PV
CD
PBM
0 h 6 h 12 h 24 h 36 h 48 h 72 h
20E (pg/female)
0 d 4 d
PV PBM
Aa
β
FTZ-F1A
20
40
60
80
100
120
a
b
ab
b

b
ab
ab
ab
0
Aa
β
FTZ-F1B
0
5
10
15
20
25
a
a
a
a
a
a
a
a
Fig. 3. (Continued).
Nuclear receptors in Aedes aegypti J. Cruz et al.
1240 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 6 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 sec-
ond experiment, the fat bodies were incubated in media
supplemented with 20E for 4 h, washed and then incu-
bated 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 1.
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
Relative mRNA (+SEM)
Relative mRNA (+SEM)
AaActin
0
50
100
150

200
250
ab
a
b
b
b
b
ab
ab
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
PV PBM
1 d 4 d 6 h 12 h 24 h 36 h 48 h 72 h
PV PBM
AaTll
0
200
300
400
a
b
b
b
b
b
b
b
AaPNR-like
0
20

40
60
80
100
120
140
a
a
a
a
a
a
a
a
10
20
AaSvp
0
20
40
60
80
a
a
a
a
a
a
a
a

AaHR38
0
20
40
60
80
100
120
140
a
a
a
a
a
a
a
a
AaS7
0
2
4
6
8
10
12
14
16
b
a
b

b
b
b
b
b
PV
EF
PBM
0 h 6 h 12 h 24 h 36 h 48 h 72 h
20E (pg/female)
50
100
150
200
250
20E (pg/female)
50
100
150
200
250
0 d 4 d0 h 6 h 12 h 24 h 36 h 48 h 72 h0 d 4 d
PV PBM
Fig. 3. (Continued).
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1241
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 8 h delay

if compared with the previously published pattern [48].
By 36 h PBM, AaEcRA levels had declined signifi-
cantly, 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 per-
iod (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 Mar-
gam 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 pre-
and vitellogenic periods, with a slight increase begin-
ning at the end of the vitellogenic period (48–72 h;
Fig. 3A). Previous 20E transcriptional regulation ana-
lysis in this laboratory, using semi-quantitative RT-
PCR, 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 tran-
scripts 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 vitello-
genesis [13], suggest a lack of significant transcriptional
and translational regulation of the two components
that make up the ecdysone receptor. Moreover,
co-immunoprecipitation experiments using nuclear
extracts of vitellogenic fat bodies of A. aegypti demon-
strated that the formation of the heterodimer AaE-
cR ⁄ 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 hetero-
dimerization with AaEcR, and, consequently, the 20E-
dependent 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–pro-
tein 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 abun-
dance 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 tran-
script profiles of AaHR3 and AaHR4 is that AaHR4 is
significantly abundant in newly eclosed female fat
bodies (pre-vitellogenic 1 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
Nuclear receptors in Aedes aegypti J. Cruz et al.
1242 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
Relative mRNA (SEM)
Relative mRNA (SEM)
0
2
4
6
8
10
12
14
16
18
**
AaUSPA
0 h
CMCM 20E Chx
20E + Chx

6 h
0
2
4
6
8
10
12
14
16
**
AaEcRA
0 h
CMCM 20E CM
4 h20E + CM
4 h
20E
16 h
0
200
400
600
800
1000
1200
1400
1600
1800
AaEcRB
*

AaEcRB
0
200
400
600
800
1000
1200
1400
1600
1800
AaUSPA
0
2
4
6
8
10
12
14
16
18
25
20
10
15
0
5
AaUSPB
**

0
2
4
6
8
10
12
14
16
AaEcRA
A
**
*
25
20
10
15
5
AaUSPB
***
0
5
4
2
3
0
1
6
AaE75A
**

**
5
4
2
3
0
1
6
AaE75A
**
****
Fig. 4. Effect of 20E on the transcription of A. aegypti nuclear receptors (NRs) in isolated pre-vitellogenic (PV) fat bodies. In the first experi-
ment (left panel), PV fat bodies dissected from female mosquitoes (5 days) were incubated in culture media (CM) for 30 min and established
as initial time point (0 h); then were incubated for 6 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), 5 day PV fat bodies were incubated in CM for 30 min and established as initial time point
(0 h); then were incubated in CM or with 20E for 4 and 16 h, or with a pulse treatment of 20E for 4 h then removal of 20E, followed by incu-
bation 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 lev-
els 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 0 h CM using Dunnett’s method. Asterisks indicate statistically significant differ-
ences at ****P £ 0.0005; ***P £ 0.005; **P £ 0.05; *P £ 0.1 levels.
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1243
that MsE75A represses the induction of MsHR3 by
binding to the consensus monomeric response element

(A ⁄ T-AGGTCA) present in its promoter [55]. Further-
more, 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
dsRNA [57]; these authors suggested a cascade activa-
tion 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 transcrip-
tional cascade described for these NRs displays some
differences from that observed for M. sexta but,
Relative mRNA (SEM)
Relative mRNA (SEM)
0 h
CMCM 20E Chx
20E + Chx
6 h
0 h
CMCM 20E CM
4 h 20E + CM
4 h
20E
16 h
AaHR39
0
2
4

6
8
10
0
1
2
3
4
5
6
AaHR78
**
AaHR78
0
1
2
3
4
5
6
0
2
4
6
8
10
AaHR39
****
*
0

200
400
600
800
1000
1200
1400
AaE78
**
*
AaE78
0
200
400
600
800
1000
1200
1400
**
AaHR4
0
2
1
13
14
0
2
4
6

8
10
12
14
AaHR4
***
0
2
4
6
8
AaHR3
B
****
AaHR3
0
2
4
6
8
**
**
Fig. 4. (Continued).
Nuclear receptors in Aedes aegypti J. Cruz et al.
1244 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 expres-

sion 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 forma-
tion, thus facilitating the induction of the DmbFTZ-
F1 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
Relative mRNA (SEM)
Relative mRNA (SEM)
0 h
CMCM 20E Chx
20E + Chx
6 h 0 h
CMCM 20E CM
4 h 20E + CM
4 h
20E
16 h
0
20
40
60
80
100
120
140

****
***
Aa
β
FTZ-F1A
C
0
10
20
30
40
50
**
AaHNF4A
0
40
80
120
160
200
AaHNF4C
Aa
β
FTZ-F1A
0
20
40
130
140
**

Aa
β
FTZ-F1B
0
100
200
300
400
***
***
AaHNF4A
0
10
20
30
40
50
AaHNF4B
0
2
4
6
8
10
12
*
AaHNF4C
0
40
80

120
160
200
0
100
200
300
400
Aa
β
FTZ-F1B
**
*
0
2
4
6
8
10
12
AaHNF4B
*
Fig. 4. (Continued).
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1245
the ecdysteroid-induced transcription factors. Such cas-
cades will likely differ among species, or even physio-
logical processes. In A. aegypti, the discrete expression
of both AaHR3 and AaHR4 suggests their function in
the regulatory hierarchy in the mosquito fat body dur-

ing 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, unpub-
lished observation), suggesting a similar regulation of
this NR as reported for other insect species. Further-
more, 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 eclo-
sion 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 dur-
ing 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 bind-
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 man-
ifested 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-depen-
dent 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 coin-
cides 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 dis-
played different expression profiles and are both regu-
lated by 20E in a similar manner. As shown in
Fig. 3C, the level of AabFTZ-F1A transcript abun-
dance was significantly higher than that of AabFTZ-
F1B in the fat bodies of newly eclosed females. After
Nuclear receptors in Aedes aegypti J. Cruz et al.
1246 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
the onset of vitellogenesis, AabFTZ-F1A dropped dra-
matically, remaining at low levels until vitellogenesis
was complete (36–72 h PBM). AabFTZ-F1B mRNA
displayed near background levels of transcript abun-
dance 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 lev-
els 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 ‘compe-
tence’ 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. melanog-
aster, where the acquisition of competence is regulated
by 20E, in A. aegypti this preparatory period is regu-
lated 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 com-
binatorial code required for specific gene activation
by 20E.
By contrast to AabFTZ-F1A, AabFTZ-F1B dis-
played a different response to 20E. Withdrawal of 20E
from the fat body culture after an initial 4 h incuba-
tion in the presence of 20E resulted in a considerable
elevation of AabFTZ-F1B mRNA (Fig. 4C), as previ-
ously 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
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 pro-
vides 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 vitello-
genic 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 iso-
forms 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 per-
iod, 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 4 days after eclosion, and quickly
dropped after a blood meal, beginning to rise again
after vitellogenesis (36–72 h PBM; Fig. 3D). Not sur-
prisingly, 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, 6 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 hepa-
tocyte differentiation and lipid homeostasis [84,85].
Mutations in the human HNF-4a cause a type II dia-
betes called maturity-onset diabetes of the young, sub-
type 1 (MODY1), which is associated with defective
glucose-dependent insulin secretion from pancreatic
beta cells [86]. HNF-4a activates the insulin gene
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1247
directly [87], but also plays a crucial role in the tran-
scriptional 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 downreg-
ulated 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 upregula-
ted 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 upregulat-
ed with a 4 h ecdysone pulse, placing it as a secondary
20E response gene; AaHNF-4B is only upregulated in
the presence of Chx, suggesting a transcript stabiliza-
tion 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 dis-
played in AaHNF-4A in hormone-free media that har-
bors 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). Address-
ing 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 mos-
quito 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 mobili-
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 do
not bind to estrogen or other natural ligands [95]. The
GAL4-LBD system showed that the LBD of Drosoph-
ila ERR displayed a remarkable switch in activity dur-
ing 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 sug-
gested 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 acetyl-
CoA. This family of NRs act synergistically with the
peroxisome proliferator-activated receptor c coactiva-
tor (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 4 pre-vitellogenesis, and remained low during
the whole vitellogenic period, with only small non-sig-
nificant 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 treat-
ment 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 con-
trolling terminal genes that result in normal develop-
ment 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 verte-
brate ortholog of Tll, is required for the correct devel-
opment 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 reg-
ulation 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 devel-
oping 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
Nuclear receptors in Aedes aegypti J. Cruz et al.
1248 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 pre-
viously discussed for AaHR51 and AaDsf.
AaSvp transcript abundance was high in the fat
body of newly eclosed females, although not signifi-
cantly different from that at other time points exam-
ined (Fig. 3F), in agreement with previous reports for
AaSvp [103]. AaHR38 mRNA also displayed its maxi-
mum 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, nei-
ther 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 over-
view 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 dis-
ease vector because it requires host contact. This
understanding of the molecular regulation of vitello-
genesis 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 ( />using the BLASTP tool against the predicted protein data-
set of A. aegypti. The sequences gleaned from the
A. aegypti database were examined manually through mul-
tiple 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
erroneous exon predictions relative to their orthologs in
other species. To address this issue, we validated our pre-
dictions 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 diffe-
rent sources: GenBank ( for
D. melanogaster, Ap. mellifera and Homo sapiens, Vector-
Base ( for Anopheles gambiae,
and Beetlebase ( />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 esti-
mate 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]. Lar-
vae 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 RNeasy
Ò
mini kit columns (Qiagen, Valencia,
CA, USA), which included an on-column DNase I diges-
tion (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 ali-
quoted; to this, cDNA of the different tissues and time
points was added. The samples were subjected to PCR
J. Cruz et al. Nuclear receptors in Aedes aegypti
FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS 1249
amplification with a number of cycles within the linear
range of amplification, preincubation at 95 °C for 15 min
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 5 min.
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 concentra-
tion = 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 Tukey-
Kramer 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-vitello-
genic 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 min in culture medium without hormone, followed

by a 6 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 6 h. A
second experiment addressed the effect of an initial induc-
tion by 10
)6
m 20E followed by removal of the said hor-
mone; this was accomplished by first providing 10
)6
m 20E
for 4 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 compara-
ble control was the maintenance of fat bodies for 16 h in
media with or without 10
)6
m 20E. As a control for all
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 normalized against ribo-
somal protein S7 transcripts, and the normalized transcripts

compared against a 0-h fat body preparation using
Dunnett’s Method (P £ 0.05) (jmp statistical discovery
software from SAS Institute).
Acknowledgements
This work was supported by National Institutes of
Health grant RO1 AI-36959. Josefa Cruz is a recipient
of a post-doctoral research grant from Department
d’Universitats, Recerca i Societat de la Informacio de
la Generalitat de Catalunya.
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Supporting information
The following supplementary material is available:
Table S1. NRs of Drosophila melanogaster, Aedes
aegypti, Anopheles gambiae, Apis mellifera and
Tribolium castaneum.
Table S2. Primers used in RT-PCR and real-time
PCR.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Nuclear receptors in Aedes aegypti J. Cruz et al.
1254 FEBS Journal 276 (2009) 1233–1254 ª 2009 The Authors Journal compilation ª 2009 FEBS