As the first step toward analysis of this gene family, wehave established their expression within the two main reproductive tissues of adult female mosquitoes: fat body and ovary.. Before
Trang 1Annotation, hormonal regulation and expression profiling
Josefa Cruz*, Douglas H Sieglaff*, , Peter Arensburger, Peter W Atkinson and Alexander S RaikhelDepartment 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 (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
Anophe-line mosquitoes are the vectors of malaria, whereas
Aedesspecies 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 standing of mosquito reproductive biology is animportant component in developing novel strategiesfor use in the control of mosquito-borne disease.Egg maturation in Aedes aegypti adult femalesincludes a process termed vitellogenesis, which involvesmassive production of yolk protein precursors (YPPs)
under-by the fat body and their subsequent internalizationinto the developing oocyte, helping to support laterembryonic 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
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 eases of humans and domestic animals Understanding the molecularmechanisms regulating mosquito egg development may contribute signi-ficantly to the development of novel vector-control strategies Previousstudies have shown that in the yellow fever mosquito Aedes aegypti, nuclearreceptors (NRs) play a key role in the endocrine regulation of reproduction.However, many mosquito NRs remain uncharacterized, some of which mayplay an important role in mosquito reproduction Publication of the genome
dis-of A aegypti allowed us to identify all NRs in this mosquito based on theirphylogenetic relatedness to those within Insecta We have determined thatthere are 20 putative A aegypti NRs, some of which are predicted to havedifferent isoforms As the first step toward analysis of this gene family, wehave established their expression within the two main reproductive tissues
of adult female mosquitoes: fat body and ovary All NR transcripts arepresent in both tissues, most displaying dynamic expression profiles duringreproductive cycles Finally, in vitro assays with isolated fat bodies wereconducted to identify the role of the steroid hormone 20-hydroxyecdysone
in modulating the expression of A aegypti NRs These data which describethe identification, expression and hormonal regulation of 20 NRs in theyellow fever mosquito lay a solid foundation for future studies on thehormonal 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.
Trang 2hormones 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,
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 mum, after which they sharply decline, concludingwith the termination of vitellogenesis In this termina-tion process, mosquito Seven-up (AaSvp), a NR mem-ber, plays a central role replacing AaUSP in theAaEcR⁄ AaUSP heterodimer complex, thereby block-ing the action of 20E [13] Another A aegypti NR,AaHNF-4c, has also been proposed to promote thetermination of Vg expression [17], but the mechanism
maxi-is still unknown The regulation of AaVg geneexpression by 20E acts not only through members ofthe NR family, but also through other transcriptionfactors 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 areadditional NRs that remain uncharacterized in themosquito, some of which may play an important role
in its reproduction In this study, we identified andbegan to characterize all putative NRs of A aegypti
We report the annotation of 19 canonical NR familymembers along with one member of the so-calledKnirps group In addition, we determined the expres-sion profiles for transcripts of these NRs within tworeproductive tissues of the adult female mosquito – thefat body and ovaries Furthermore, using an in vitrofat body culture system allowed us to identify NRsresponsive to 20E This work provides a foundationfrom which future studies in post-genomic functionalanalysis of NR developmental regulation in mosqui-toes can begin
Results and DiscussionIdentification of NRs in the genome of A aegypti
We identified A aegypti NRs from the 1.0 Genebuildassembly of the A aegypti genome (created from amerge of the TIGR and VectorBase 0.5 annotationsets) Protein homology searches were performed usingindividual members of the NR family from D mela-nogaster compared against the A aegypti database(http://aaegypti.vectorbase.org/index.php) We identi-fied 20 NR family members in the A aegypti genome,
19 of which are likely orthologs of D melanogasterNRs, 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 bled automatic predicted sequences in the A aegyptigenomic database; with some given splice sites andexons not predicted in our original manual annotation
Trang 3unassem-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 asAabFTZ-F1A (previously named AabFTZ-F1) [23]and AabFTZ-F1B
Phylogenetic analysis
We conducted a phylogenetic analysis of the NRs fromthe five insect genomes sequenced so far: D melano-gaster, Anopheles gambiae, Ap mellifera, T castaneumand A aegypti as well as the sequences of the humanorthologs When different isoforms were recovered,only the longest amino acid sequences that includedthe DNA binding, hinge and ligand-binding domainswere 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.
NCBI accession no.
20E response
AAEL002768
Trang 4this 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 (seeTable S2 for primer sequences) Two biological repli-cates were analyzed, and Fig 2 depicts the profilematching both replicates
An increase in transcript abundance for AaEcRA,
AaHR4, AaE78 and AaHR39 occurred in both tissues,correlating with the known rise in ecdysteroids in vivo,whereas AaUSP-B, AaHR38, AaTll and AaPNR-likeonly displayed an increase in transcript abundance inthe fat body during this same period (Fig 2) This sug-gests that these later orphan NRs may be hormoneinducible, which we addressed using in vitro assays(see below) Many NR transcripts displayed a decreaseduring the initial phase of vitellogenesis (6 + 12 hPBM) 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) werealso analyzed in our in vitro assay Any kind of fluctu-ation in expression levels in the developmental timepoint 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 abetter clarification of their developmental expressionpatterns, we increased the number of PCR cycles forboth fat body and ovary samples for many NR tran-scripts studied Such an effort did not lead to betterresolution (not shown)
In D melanogaster, DmDsf is expressed in a group
of neurons in the central nervous system and isrequired for normal sexual behavior [32] FAX-1, theortholog of HR51 and HR83 in C elegans, is requiredfor neurotransmitter expression in specific interneurons[33], and the human ortholog, PNR, displays a highlyrestricted expression in retinal tissues [34] These stud-ies imply a tissue-restricted expression of the members
of this NR family, suggesting that what we observedmay have simply been basal level expression, not dis-cernable using our RT-PCR analysis By contrast tothis restricted expression observed within nervoussystems, HR96 has been implicated in regulating xeno-biotic responses in D melanogaster and is highlyexpressed in tissues that monitor and metabolize xeno-biotics, including the fat body [35] It has also beenestablished that HR96 is broadly expressed throughoutlarval development and metamorphosis [7] Given theexpression 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 areexpressed at a constant level within the ovaries duringthe period analyzed However, AaE75A, AaE75B,
Trang 5Fig 1 Phylogenetic tree of insect NRs The different NR families are organized into groupings, NR1–NR6 The tree was constructed ing the distance-based neighbor-joining method, using the NRs sequences of D melanogaster (Dm), A aegypti (Aa), An gambiae (Ag),
follow-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.
Trang 6AaE75C 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, 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 andmetamorphosis in insects, its potential role in promot-ing oogenesis has received significantly less attention.EcR mutant females of D melanogaster displayabnormal 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 areduction in the number of follicular cells in the basaloocyte, subsequent nymphal developmental defectsand even a reorganization of the follicular epithelium
in the resulting adults [40] DmE75A and DmE75Bhave 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 transitionfrom vitellogenesis to choriogenesis [43] Although thepresence of dynamic expression profiles of various
NR family members within Insecta suggests the ative use of ecdysone-regulatory hierarchies in fatbody and ovary reproductive functions throughoutthis class, the complexity of the meroistic ovaries ofmosquitoes makes extrapolation of function for theseNRs from the condition in other insects very difficult.That is, each ovariole in mosquitoes consists of agermline-derived oocyte and nurse cells surrounded bysomatically derived follicle cells; with our methodol-ogy, we are unable to distinguish among these threedistinctive cell types [44] Thus, the NR transcriptspresent in our ovary samples could either be involved
reiter-in ovary development or comprise a maternal bution for later embryonic development Indeed, nine
contri-NR transcripts are maternally loaded in D aster ovaries [7]
melanog-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 qPCRagainst 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 ofthree independent collections of mosquito femalesstaged at different time points during pre-vitellogenicand vitellogenic stages The same amount of RNA wasretro-transcribed and analyzed by means of qPCRusing specific primer pairs for each NR (Table S2)
In vivo fat body NR transcript expression levels werestandardized by total RNA input, because the fat body
is a dynamically developing tissue both before andfollowing 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.
Trang 70 500 1000 1500 2000 2500
b
ab a a
AaHR4
0
300 1000 1500 2000 2500
a
a a
0
100 200
50 100 150 200 250
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).
Trang 8‘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 observedthrough 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 acondition 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
10
AaHNF4B
0 2000 4000 6000 8000
10 000
b a
ab a
AaHNF4C
0 500 1000 1500 2000 2500 3000 3500
a a
AaHNF4A
0 1000 2000 3000 4000 5000
b
a a
Fig 3 (Continued).
Trang 9same developmental stage, hence the use of ribosomal
protein S7transcripts 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 ond experiment, the fat bodies were incubated in mediasupplemented with 20E for 4 h, washed and then incu-bated for 12 more hours in a hormone-free medium
sec-As a control, fat bodies were incubated with or without20E RNA extracted from these samples was analyzed
by means of qPCR, and transcripts were normalizedover AaS7 A summary of the 20E inducibility of thetranscripts analyzed is presented in Table 1
The A aegypti ecdysone receptorTwo EcR isoforms (AaEcRA and AaEcRB) and twoUSP isoforms (AaUSP-A and AaUSP-B) have beencharacterized previously in A aegypti [48–50] The
Relative mRNA (+SEM) Relative mRNA (+SEM
AaActin
0 50 100 150 200 250
ab
a
ab ab
a
AaHR38
0 20 40 60 80 100 120 140
Trang 10expression 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 nuclearextracts of vitellogenic fat bodies of A aegypti demon-strated that the formation of the heterodimer AaE-
cR⁄ AaUSP can be regulated by other NRs throughprotein–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⁄ AaUSPheterodimer [13], indicating that the regulation of theactivity of these proteins occurs through protein–pro-tein interactions, as well as ligand-mediated switch
NR transcripts regulated by 20EThe Aedes E75 NR family has three isoforms, eachwith a distinctive N-terminal A⁄ B domain [36].AaE75B cannot bind DNA due to the lack of one ofits zinc fingers All three isoforms present a similarpattern of expression and regulation by 20E [36]; thus,
we only present the data corresponding to the AaE75Aisoform As in D melanogaster [51] and Manduca sexta[52], AaE75A is expressed transiently very early in theecdysone-induced regulatory cascade and is directlyregulated by 20E [36], response characteristics thatdefine it as an early-gene
Expression of AaHR3 and AaHR4 occurred onlyafter that of AaE75A reached its peak (Fig 3B).AaHR3 exhibited a sharp increase in transcript abun-dance peaking at 24 h PBM [37], whereas AaHR4mRNA levels remained much more flat (Fig 3B) In
in vitro fat body culture, AaHR3 transcription wasactivated by 20E, but this upregulation only becamesignificant if the tissue was incubated simultaneouslywith 20E and Chx, or was continuously exposed to thehormone for 16 h [37] AaHR4 transcription wassignificantly upregulated by combining 20E and Chx inthe culture medium (Fig 4B), but the presence of20E alone also increased the transcript abundance,although not significantly (Fig 4B)
The primary difference between the observed script profiles of AaHR3 and AaHR4 is that AaHR4 issignificantly abundant in newly eclosed female fatbodies (pre-vitellogenic 1 day, Fig 3B), suggesting apossible role in the transition between pupae andadult It is generally believed that gene transcriptsinduced in vitro only in the presence of 20E andthe protein synthesis inhibitor Chx, as observed forAaHR4 and AaHR3, are negatively repressed by20-induced genes (e.g the early response genes) [53,54]
tran-M sexta GV1 cell transfection assays demonstrated
Trang 11Relative mRNA (SEM) Relative mRNA (SEM) 0
16
**
AaEcRA
0 h CM
AaUSPA
0 2 4 6 8 10 12 14 16 18
5
0
5 4
2 3
0 1